Exhibit Growth Defects Research Articles

SAGA1 and MITH1 produce matrix-traversing membranes in the CO2-fixing pyrenoid.

Approximately one-third of global CO2 assimilation is performed by the pyrenoid, a liquid-like organelle found in most algae and some plants. Specialized pyrenoid-traversing membranes are hypothesized to drive CO2 assimilation in the pyrenoid by delivering concentrated CO2, but how these membranes are made to traverse the pyrenoid matrix remains unknown. Here we show that proteins SAGA1 and MITH1 cause membranes to traverse the pyrenoid matrix in the model alga Chlamydomonas reinhardtii. Mutants deficient in SAGA1 or MITH1 lack matrix-traversing membranes and exhibit growth defects under CO2-limiting conditions. Expression of SAGA1 and MITH1 together in a heterologous system, the model plant Arabidopsis thaliana, produces matrix-traversing membranes. Both proteins localize to matrix-traversing membranes. SAGA1 binds to the major matrix component, Rubisco, and is necessary to initiate matrix-traversing membranes. MITH1 binds to SAGA1 and is necessary for extension of membranes through the matrix. Our data suggest that SAGA1 and MITH1 cause membranes to traverse the matrix by creating an adhesive interaction between the membrane and matrix. Our study identifies and characterizes key factors in the biogenesis of pyrenoid matrix-traversing membranes, demonstrates the importance of these membranes to pyrenoid function and marks a key milestone toward pyrenoid engineering into crops for improving yields.

Biogenesis, engineering and function of membranes in the CO 2 -fixing pyrenoid.

Approximately one-third of global CO 2 assimilation is performed by the pyrenoid 1 , a liquid-like organelle found in most algae and some plants 2 . Specialized membranes are hypothesized to drive CO 2 assimilation in the pyrenoid by delivering concentrated CO 2 3,4 , but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana . SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO 2 -limiting conditions, providing evidence for the function of membrane tubules in CO 2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

Histone H3 E50K mutation confers oncogenic activity and supports an EMT phenotype.

Sequencing of human patient tumors has identified recurrent missense mutations in genes encoding core histones. We report that mutations that convert histone H3 amino acid 50 from a glutamate to a lysine (H3E50K) support an oncogenic phenotype in human cells. Expression of H3E50K is sufficient to transform human cells as evidenced by a dramatic increase in cell migration and invasion, and a statistically significant increase in proliferation and clonogenicity. H3E50K also increases the invasive phenotype in the context of co-occurring BRAF mutations, which are present in patient tumors characterized by H3E50K. H3E50 lies on the globular domain surface in a region that contacts H4 within the nucleosome. We find that H3E50K perturbs proximal H3 post-translational modifications globally and dysregulates gene expression, activating the epithelial to mesenchymal transition. Functional studies using S. cerevisiae reveal that, while yeast cells that express H3E50K as the sole copy of histone H3 show sensitivity to cellular stressors, including caffeine, H3E50K cells display some genetic interactions that are distinct from the characterized H3K36M oncohistone yeast model. Taken together, these data suggest that additional histone H3 mutations have the potential to be oncogenic drivers and function through distinct mechanisms that dysregulate gene expression.

Transfer RNA acetylation regulates in vivo mammalian stress signaling.

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

Overlapping coactivator function is required for transcriptional activation by the Candida glabrata Pdr1 transcription factor.

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and 2 different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and 2 different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high-level Pdr1-dependent gene transcription.

SNF1 plays a crucial role in the utilization of n-alkane and transcriptional regulation of the genes involved in it in the yeast Yarrowia lipolytica

Yarrowia lipolytica is an ascomycetous yeast that can assimilate hydrophobic carbon sources including oil and n-alkane. The sucrose non-fermenting 1/AMP-activated protein kinase (Snf1/AMPK) complex is involved in the assimilation of non-fermentable carbon sources in various yeasts. However, the role of the Snf1/AMPK complex in n-alkane assimilation in Y. lipolytica has not yet been elucidated. This study aimed to clarify the role of Y. lipolytica SNF1 (YlSNF1) in the utilization of n-alkane. The deletion mutant of YlSNF1 (ΔYlsnf1) exhibited substantial growth defects on n-alkanes of various lengths (C10, C12, C14, and C16), and its growth was restored through the introduction of YlSNF1. Microscopic observations revealed that YlSnf1 tagged with enhanced green fluorescence protein showed dot-like distribution patterns in some cells cultured in the medium containing n-decane, which were not observed in cells cultured in the medium containing glucose or glycerol. The RNA sequencing analysis of ΔYlsnf1 cultured in the medium containing n-decane exhibited 302 downregulated and 131 upregulated genes compared with the wild-type strain cultured in the same medium. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that a significant fraction of the downregulated genes functioned in peroxisomes or were involved in the metabolism of n-alkane and fatty acids. Quantitative real-time PCR analysis confirmed the downregulation of 12 genes involved in the metabolism of n-alkane and fatty acid, ALK1–ALK3, ALK5, ADH7, PAT1, POT1, POX2, PEX3, PEX11, YAS1, and HFD3. Furthermore, ΔYlsnf1 exhibited growth defects on the medium containing the metabolites of n-alkane (fatty alcohol and fatty aldehyde). These findings suggest that YlSNF1 plays a crucial role in the utilization of n-alkane in Y. lipolytica. This study provides important insights into the advanced biotechnological applications of this yeast, including the bioconversion of n-alkane to useful chemicals and the bioremediation of petroleum-contaminated environments.

Overlapping coactivator function is required for transcriptional activation by the Candida glabrata Pdr1 transcription factor.

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1 . Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification (TAP)-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and two different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and two different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high level Pdr1-dependent gene transcription.

A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers.

Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.

A chlorophyll c synthase widely co-opted by phytoplankton.

Marine and terrestrial photosynthesis exhibit a schism in the accessory chlorophyll (Chl) that complements the function of Chl a: Chl b for green plants versus Chl c for most eukaryotic phytoplankton. The enzymes that mediate Chl c biosynthesis have long remained elusive. In this work, we identified the CHLC dioxygenase (Phatr3_J43737) from the marine diatom Phaeodactylum tricornutum as the Chl c synthase. The chlc mutants lacked Chl c, instead accumulating its precursors, and exhibited growth defects. In vitro, recombinant CHLC protein converted these precursors into Chl c, thereby confirming its identity. Phylogenetic evidence demonstrates conserved use of CHLC across phyla but also the existence of distinct Chl c synthases in different algal groups. Our study addresses a long-outstanding question with implications for both contemporary and ancient marine photosynthesis.

Chemical Genetics Screen of EVP4593 Sensitivity in Budding Yeast Identifies Effects on Mitochondrial Structure and Function.

Mitochondria are essential eukaryotic organelles. Mitochondrial dysfunction can lead to mitochondrial myopathies and may contribute to neurodegenerative diseases, cancer, and diabetes. EVP4593, a 6-aminoquinazoline derivative with therapeutic potential, has been shown to inhibit NADH-ubiquinone oxidoreductase (Complex I) of the mitochondrial electron transport chain, causing the release of reactive oxygen species (ROS) and a reduction in ATP synthesis. In isolated mitochondria, EVP4593 inhibits respiration in the nanomolar range (IC 50 = 14-25 nM). However, other EVP4593-specific effects on biological processes have also been described. Consistent with an effect on mitochondrial function in budding yeast, we find that EVP4593 [>25µM] induces a pronounced growth defect when wildtype cells are grown on a non-fermentable carbon source. This sensitivity to EVP4593 is exacerbated by deletion of PDR5 , an ABC transporter that confers multidrug resistance. To better understand the cellular pathways and processes affected by EVP4593, we conducted a genome-wide chemical genetics screen of the yeast knockout collection. The objective was to identify yeast gene deletion strains that exhibit growth defects when subjected to a sublethal concentration of EVP4593 [15µM]. Our screen identified 21 yeast genes that are required for resistance to 15µM EVP4593 in glycerol-containing media. The genes identified in our screen are functionally involved in several distinct categories including mitochondrial structure and function, translational regulation and nutritional sensing, cellular stress response and detoxification. Additionally, we identified cellular phenotypes associated with the exposure to EVP4593, including changes in mitochondrial structure. In conclusion, our study represents the first genome-wide screen in yeast to identify the genetic pathways and cell-protective mechanisms involved in EVP4593 resistance and reveals that this small molecule inhibitor affects both mitochondrial structure and function.

Role of Staphylococcus aureus Formate Metabolism during Prosthetic Joint Infection.

Biofilms are bacterial communities characterized by antibiotic tolerance. Staphylococcus aureus is a leading cause of biofilm infections on medical devices, including prosthetic joints, which represent a significant health care burden. The major leukocyte infiltrate associated with S. aureus prosthetic joint infection (PJI) is granulocytic myeloid-derived suppressor cells (G-MDSCs), which produce IL-10 to promote biofilm persistence by inhibiting monocyte and macrophage proinflammatory activity. To determine how S. aureus biofilm responds to G-MDSCs and macrophages, biofilms were cocultured with either leukocyte population followed by RNA sequencing. Several genes involved in fermentative pathways were significantly upregulated in S. aureus biofilm following G-MDSC coculture, including formate acetyltransferase (pflB), which catalyzes the conversion of pyruvate and coenzyme-A into formate and acetyl-CoA. A S. aureus pflB mutant (ΔpflB) did not exhibit growth defects in vitro. However, ΔpflB formed taller and more diffuse biofilm compared to the wild-type strain as revealed by confocal microscopy. In a mouse model of PJI, the bacterial burden was significantly reduced with ΔpflB during later stages of infection, which coincided with decreased G-MDSC influx and increased neutrophil recruitment, and ΔpflB was more susceptible to macrophage killing. Although formate was significantly reduced in the soft tissue surrounding the joint of ΔpflB-infected mice levels were increased in the femur, suggesting that host-derived formate may also influence bacterial survival. This was supported by the finding that a ΔpflBΔfdh strain defective in formate production and catabolism displayed a similar phenotype to ΔpflB. These results revealed that S. aureus formate metabolism is important for promoting biofilm persistence.

Flavobacterium columnare ferric iron uptake systems are required for virulence.

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.

Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

Mono-methylation of the fourth lysine on the N-terminal tail of histone H3 was found to support the induction of RNA polymerase II transcription in S. cerevisiae during nutrient stress. In S. cerevisiae, the mono-, di- and tri-methylation of lysine 4 on histone H3 (H3K4) is catalyzed by the protein methyltransferase, Set1. The three distinct methyl marks on H3K4 act in discrete ways to regulate transcription. Nucleosomes enriched with tri-methylated H3K4 are usually associated with active transcription whereas di-methylated H3K4 is associated with gene repression. Mono-methylated H3K4 has been shown to repress gene expression in S. cerevisiae and is detected at enhancers and promoters in eukaryotes. S. cerevisiae set1Δ mutants unable to methylate H3K4 exhibit growth defects during histidine starvation. The growth defects are rescued by either a wild-type allele of SET1 or partial-function alleles of set1, including a mutant that predominantly generates H3K4me1 and not H3K4me3. Rescue of the growth defect is associated with induction of the HIS3 gene. Growth defects observed when set1Δ cultures were starved for isoleucine and valine were also rescued by wild-type SET1 or partial-function set1 alleles. The results show that H3K4me1, in the absence of H3K4me3, supports transcription of the HIS3 gene and expression of one or more of the genes required for biosynthesis of isoleucine and valine during nutrient stress. Set1-like methyltransferases are evolutionarily conserved, and research has linked their functions to developmental gene regulation and several cancers in higher eukaryotes. Identification of mechanisms of H3K4me1-mediated activation of transcription in budding yeast will provide insight into gene regulation in all eukaryotes.

Ablation of Kex2 activity enhances proIAPP proteotoxicity in yeast

Pancreatic deposition of Islet Amyloid PolyPeptide (IAPP) is a histopathological hallmark of type 2 diabetes. Inadequate processing of immature IAPP by proprotein convertases (PCs) leads to the accumulation of unprocessed forms, which in turn favors increased aggregate formation in pancreatic β-cells. Kexin 2 (Kex2) of Saccharomyces cerevisiae is the prototype of eukaryotic PCs family, but to date no direct correlations between Kex2 activity and preproIAPP processing in yeast have been reported. In this study we aimed to address the possible role of Kex2 on IAPP maturation as a tool to investigate the contribution of impaired processing towards IAPP proteototoxicity. Genetically modified S. cerevisiae models lacking the KEX2 gene and carrying different chimeric fusions of human IAPP linked to GFP were used. The cytotoxic effects of Kex2 ablation were assessed by means of growth curve analysis and cell viability assays using flow cytometry. IAPP protein profile was evaluated by immunoblotting assays using an anti-IAPP antibody. Intracellular IAPP aggregates were monitored by confocal fluorescence microscopy. Data showed that kex2 mutants exhibit growth defects, potentiated by preproIAPP-GFP, proIAPP-GFP and mature IAPP-GFP expression with an increased cytotoxicity for proIAPP-GFP. Notwithstanding, Kex2 absence does not seem to affect IAPP protein pattern nor the frequency/distribution of intracellular IAPP aggregates in yeast. Our findings suggest that Kex2 is not essential for IAPP processing in yeast, at least under the conditions tested. Keywords: Amylin, Islet Amyloid Polypeptide (IAPP), Kexin 2, Prohormone processing, Saccharomyces cerevisiae

Integrated mRNA and miRNA Profiling Revealed Deregulation of the Cellular Stress Response in Bone Marrow Mesenchymal Stem Cells Derived from Patients with Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet destruction and decreased platelet production. Our understanding of its pathogenesis remains limited due to the complexity of the immune system and the heterogeneity of the disease (Rivière et al. Br J Haematol. 2015, 170). Recently, we found that the bone marrow mesenchymal stem cells (MSCs) derived from ITP patients (MSC-ITP) exhibited growth defects and functional abnormalities, which may be implicated in the breakdown of self-tolerance in ITP (Zhang et al. Stem Cells Transl Med.2016, 5). The underlying mechanism of these defects remains unclear. In this study, by utilizing microarray technique and bioinformatics analysis, we aimed to profile both the mRNA and microRNA expression and decipher the underlying mechanism involved in the impairment of MSC-ITP.Methods: MSCs were derived from 4 ITP patients and age-, sex- and BMI-matched healthy individuals served as control. Microarray was performed on total RNA specimens using Affymetrix mRNA and miRNA microarray.Results: Overall, we identified 740 genes and 32 microRNAs that were differently expressed in ITP patients compared with controls. Function and disease analysis revealed that the differentially expressed genes were most significantly enriched in the processes of cell death and survival, cellular development, cell growth and proliferation and cell cycle, which is in agreement with our previous study demonstrating the excessive apoptosis and senescence of MSCs from ITP patients. Ingenuity canonical pathway annotation enabled us to assign differentially expressed genes to 57 pathways. Most of these top enriched pathways have functions in cell proliferation, cell cycle and cell death. Of note, unfolded protein response (UPR), which is a defense response to protect cells from damage induced by endoplasmic reticulum stress, was identified as the most significantly changed. Furthermore, GSEA identified that UPR was negatively correlated with MSC-ITP. These data suggested that deficient UPR may play key roles in MSC-ITP. Upstream analysis revealed a significantly decreased overall DNA transcription, and many stress response transcriptional factors such as NUPR1 and TGF-b were down-regulated, indicating a potential inability of the cellular stress response. Further, we utilized gene network analysis in an attempt to target the key “cross-road” regulator in MSC-ITP. Network analysis demonstrated that JUN and CDKN1A were two key cross regulators with multiple roles in the biological process of MSC-ITP. JUN and CDKN1A were critical downstream regulators in UPR and the p53 pathway, respectively, implying a potential cross-talk between UPR and the p53 pathway, the latter of which has been verified in MSC-ITP in our previous study. Finally, we utilized microRNA-mRNA interactions analysis to improve our understanding of the regulated genes. Reactome enrichment of the predicted targeted genes of differentially expressed microRNA indicated that the cellular response to stress was deregulated in MSC-ITP. Then, the predicted target genes were cross-referenced against the mRNAs significantly regulated in MSC-ITP and MSC-control, resulting in 163 overlap genes. Again, UPR was identified as the most related process in MSC-ITP by Reactome enrichment, which further confirmed the association between UPR and the defect of MSC-ITP.Conclusion: Taken together, our results illustrate that the mRNA and microRNA profiles change in MSC-ITP, which revealed a weakened ability to initiate the cellular stress response to protect cells from damage and restore homeostasis. Considering MSCs as an important immunoregulator, these results in MSC-ITP may shed light on the complex ITP bone marrow niche and on the breakdown of self-tolerance. DisclosuresNo relevant conflicts of interest to declare.

Construction and investigation of plasmid-induced effects on growth of GFP-expressing Salmonella strains

Background: Bacteria such as the Salmonella species had been extensively studied not only for its mechanism of pathogenicity, but for its beneficial utilisation in bacterial-mediated tumour therapy. Genetically modified bacterial strain, BDLA Salmonella Agona (BDLA S. Agona) had shown great promise as tumour targeting and suppressing agent. By, constructing green fluorescent protein (GFP) expressing of this strain, it would be beneficial as it could be used to further study the interaction of the BDLA S. Agona strain with various cancerous cells and the host at the systemic level. However, GFP plasmid-induced strains may exhibit growth defects, and this was investigated in this study. Methods: The GFP-transformed Salmonella strains were constructed, and the replication rate of the strains were investigated using the BacterioScan™ 216R instrument. The replication rate between the strains was compared from the 24-hour data collected and analysed as growth curves. The 24-hour growth curves were constructed using the Log CFU/mL and OD650 data collected by the instrument. Results: It was observed from the growth curves that the transformation of the GFP plasmid into different Salmonella strains did not affect the replication rate of the bacteria. Discussion and Conclusion: This finding answers the objective of the study, and it was concluded that incorporation of the GFP plasmid does not cause any negative effect on bacterial growth. GFP-transformed Salmonella, specifically the BDLA S. Agona strain, could be utilised as a powerful tool for future studies on the mechanism of tumour suppression and real-time in vivo biodistribution of the strain.

Construction and investigation of plasmid-induced effects on growth of GFP-expressing Salmonella strains

Background: Bacteria such as the Salmonella species had been extensively studied not only for its mechanism of pathogenicity, but for its beneficial utilisation in bacterial-mediated tumour therapy. Genetically modified bacterial strain, BDLA Salmonella Agona (BDLA S. Agona) had shown great promise as tumour targeting and suppressing agent. By, constructing green fluorescent protein (GFP) expressing of this strain, it would be beneficial as it could be used to further study the interaction of the BDLA S. Agona strain with various cancerous cells and the host at the systemic level. However, GFP plasmid-induced strains may exhibit growth defects, and this was investigated in this study. Methods: The GFP-transformed Salmonella strains were constructed, and the replication rate of the strains were investigated using the BacterioScan™ 216R instrument. The replication rate between the strains was compared from the 24-hour data collected and analysed as growth curves. The 24-hour growth curves were constructed using the Log CFU/mL and OD650 data collected by the instrument. Results: It was observed from the growth curves that the transformation of the GFP plasmid into different Salmonella strains did not affect the replication rate of the bacteria. Discussion and Conclusion: This finding answers the objective of the study, and it was concluded that incorporation of the GFP plasmid does not cause any negative effect on bacterial growth. GFP-transformed Salmonella, specifically the BDLA S. Agona strain, could be utilised as a powerful tool for future studies on the mechanism of tumour suppression and real-time in vivo biodistribution of the strain.

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum

BackgroundThe fungicide fludioxonil over-stimulates the fungal response to osmotic stress, leading to over-accumulation of glycerol and hyphal swelling and bursting. Fludioxonil-resistant fungal strains that are null-mutants for osmotic stress response genes are easily generated through continual sub-culturing on sub-lethal fungicide doses. Using this approach combined with RNA sequencing, we aimed to characterise the effects of mutations in osmotic stress response genes on the transcriptional profile of the important agricultural pathogen Sclerotinia sclerotiorum under standard laboratory conditions. Our objective was to understand the impact of disruption of the osmotic stress response on the global transcriptional regulatory network in an important agricultural pathogen.ResultsWe generated two fludioxonil-resistant S. sclerotiorum strains, which exhibited growth defects and hypersensitivity to osmotic stressors. Both had missense mutations in the homologue of the Neurospora crassa osmosensing two component histidine kinase gene OS1, and one had a disruptive in-frame deletion in a non-associated gene. RNA sequencing showed that both strains together differentially expressed 269 genes relative to the parent during growth in liquid broth. Of these, 185 (69%) were differentially expressed in both strains in the same direction, indicating similar effects of the different point mutations in OS1 on the transcriptome. Among these genes were numerous transmembrane transporters and secondary metabolite biosynthetic genes.ConclusionsOur study is an initial investigation into the kinds of processes regulated through the osmotic stress pathway in S. sclerotiorum. It highlights a possible link between secondary metabolism and osmotic stress signalling, which could be followed up in future studies.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans.

In bacteria, DNA methylation can be facilitated by ‘orphan’ DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

Centromere deletion in Cryptococcus deuterogattii leads to neocentromere formation and chromosome fusions.

The human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate the impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9∆ mutants, cen10∆ mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37°C), the cen10∆ chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.