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Introduction: Low temperature inhibits the growth of most microorganisms. However, some microbes can grow well in a low temperature, even a freezing temperature. Methods: In this study, the mechanisms conferring cold resistance in the cryophylactic yeast Metschnikowia (M.) pulcherrima MS612, an isolate of the epidermis of ice grapes, were investigated based on comparative transcriptome analysis. Results: A total of 6018 genes and 374 differentially expressed genes (> 2-fold, p < 0.05) were identified using RNA-Seq. The differentially expressed genes were mainly involved in carbohydrate and energy metabolism, transport mechanisms, antifreeze protection, lipid synthesis, and signal transduction. M. pulcherrima MS612 maintained normal growth at low temperature (5°C) by enhancing energy metabolism, sterol synthesis, metal ion homeostasis, amino acid and MDR transport, while increased synthesis of glycerol and proline transport to improve its resistance to the freezing temperature (-5°C). Furthermore, cAMPPKA and ERAD signaling pathways contribute to resist the low temperature and the freezing temperature, respectively. Conclusion: This study provides new insights into cold resistance in cryophylactic microorganisms for maneuvering various metabolism to resist different cold environment. [ABSTRACT FROM AUTHOR]

Introduction: Tobacco vein mottling virus (TVMV) is a member of the family Potyviridae. The 3' untranslated region (3'UTR) of viral genomic RNA has been reported to significantly impact viral infection. Nevertheless, the role of the TVMV 3'UTR during viral infection remains unknown. Methods: Here, a 3'UTR-GFP expression vector was transiently expressed in Nicotiana benthamiana, in which the 3'UTR of TVMV was introduced upstream of the green fluorescent protein (GFP) gene. Transcriptome sequencing was performed to analyze the genes associated with plant resistance. The effect of the TVMV 3'UTR on GFP expression was studied using an Agrobacteriummediated transient expression assay, revealing that the TVMV 3'UTR significantly inhibited GFP expression. Transcriptome analysis of differentially expressed genes in 3'UTR-GFP in N. benthamiana was performed to elucidate the why the TVMV 3'UTR inhibited GFP expression. Results: Eighty genes related to plant disease resistance were differentially expressed, including 29 upregulated and 51 downregulated genes. Significantly upregulated genes included those encoding the calcium-binding protein CML24, leucine-rich repeat receptor-like tyrosine-protein kinase, and respiratory burst oxidase homolog protein E. The significantly downregulated genes included calcium-binding protein 7, ethylene-responsive transcription factor 10, endoglucanase 5, and receptor-like protein kinase. Discussion: These findings indicate that the 3'UTR of TVMV may inhibit the expression of GFP gene by inducing the expression of plant resistance genes. This study provides a theoretical basis for further research on the function and mechanism of the TVMV 3'UTR. [ABSTRACT FROM AUTHOR]

Introduction: The severity of Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is often dictated by a range of comorbidities. A considerable literature suggests iron deficiency and iron overload may contribute to increased infection, inflammation and disease severity, although direct causal relationships have been difficult to establish. Methods: Here we generate iron deficient and iron loaded C57BL/6 J mice by feeding standard low and high iron diets, with mice on a normal iron diet representing controls. All mice were infected with a primary SARS-CoV-2 omicron XBB isolate and lung inflammatory responses were analyzed by histology, immunohistochemistry and RNA-Seq. Results: Compared with controls, iron deficient mice showed no significant changes in lung viral loads or histopathology, whereas, iron loaded mice showed slightly, but significantly, reduced lung viral loads and histopathology. Transcriptional changes were modest, but illustrated widespread dysregulation of inflammation signatures for both iron deficient vs. controls, and iron loaded vs. controls. Some of these changes could be associated with detrimental outcomes, whereas others would be viewed as beneficial. Discussion: Diet-associated iron deficiency or overload thus induced modest modulations of inflammatory signatures, but no significant histopathologically detectable disease exacerbations. [ABSTRACT FROM AUTHOR]

Introduction: Reactive oxygen species (ROS) generation is a common disease defense mechanism in plants. However, it is unclear whether Citrus host activates defense response against Diaporthe citri causing citrus melanose disease by producing ROS, and the underlying molecular mechanisms are unknown. Methods: DAB staining and RNA-Seq technology were used to compare the active oxygen burst and differential gene expression, respectively, in uninfected and infected Citrus sinensis leaves at different time points during D. citri infection in vivo. The functions of CsRBOH (a significant DEG) were confirmed in N. benthamiana through the Agrobacterium-mediated transient expression system. Results: DAB staining indicated that C. sinensis initiated defense against D. citri infection within 24 h by generating ROS. Illumina sequencing revealed 25,557 expressed genes of C. sinensis. The most upregulated DEGs (n = 1,570) were identified 72 h after fungal inoculation (sample denoted as CD72). In the CD72 vs. Cs (samples at 0 h after fungal inoculation) comparison, the KEGG pathway category with the highest number of genes (n = 62) and most significant enrichment was Protein processing in endoplasmic reticulum, followed by Glutathione metabolism and MAPK signaling pathway-plant. GO analysis revealed that the DEGs of CD72 vs. Cs related to active oxygen burst and chitin recognition were significantly grouped into the regulation of biological processes and molecular functions, with GO terms including response to ROS, response to fungus, and oxidoreductase activity. Remarkably, CsRBOH was significantly enriched in the GO and KEGG analyses, and its expression pattern in qRT-PCR and DAB staining results were consistent. Among the 63 ROS-related DEGs, HSP genes and genes associated with the peroxidase family were highly significant as revealed by protein–protein interaction networks. Furthermore, ROS accumulation, cell death, and upregulation of defense-related genes were observed in N. benthamiana leaves with CsRBOH expressed through the Agrobacterium-mediated transient expression system. Conclusion: Our findings suggested that C. sinensis activates CsRBOH and ROSrelated genes, leading to ROS accumulation to resist the invasion by D. citri. This study laid the foundation for future research on molecular mechanisms and breeding of C. sinensis cultivars resistant to citrus melanose. [ABSTRACT FROM AUTHOR]

Metarhizium is widely known for its role as an arthropod biocontrol agent and plant bioinoculant. By using mass-production industrial methods, it is possible to produce large amounts of fungal single-celled propagules (including blastospores) to be applied in the field. However, in the environment, the solar ultraviolet components (particularly UV-B) can harm the fungus, negatively impacting its pathogenicity toward the arthropod pest. The present study is the first to use comparative genome-wide transcriptome analyses to unveil changes in gene expression between Metarhizium pingshaense blastospores exposed or not to UV-B. Relative blastospores culturability was calculated 72 h after UV-B exposure and exhibited 100% culturability. In total, 6.57% (n = 728) out of 11,076 predicted genes in M. pingshaense were differentially expressed after UV-B exposure: 320 genes (44%; 320/728) were upregulated and 408 (56%; 408/720) were downregulated in the UV-B exposed blastospores. Results unveiled differentially expressed gene sets related to fungal virulence, production of secondary metabolites, and DNA repair associated with UV damage; genes related to virulence factors were downregulated, and genes associated with nucleotide excision repair were upregulated. These findings illustrate critical aspects of Metarhizium blastospores strategies to overcome UV-B damage and survive solar radiation exposures in insulated fields.

Introduction: Sodium pheophorbide a (SPA) is a natural plant-derived photosensitizer, with high photoactivated antifungal activity against some phytopathogenic fungi. However, its fungicidal effect on Diaporthe mahothocarpus, a novel pathogen that causes Camellia oleifera leaf spot blight, is unclear. Methods: In the present study, we explored its inhibitory effects on spore germination and mycelial growth of D. mahothocarpus. Then we determined its effects on the cell membrane, mycelial morphology, redox homeostasis, and cell death through bioassay. Finally, RNA-seq was used further to elucidate its mode of action at the transcriptional level. Results: We found that SPA effectively inhibited the growth of D. mahothocarpus, with half-maximal effective concentrations to inhibit mycelial growth and spore germination of 1.059 and 2.287 mg/mL, respectively. After 1.0 mg/mL SPA treatment, the conductivity and malondialdehyde content of D. mahothocarpus were significantly increased. Scanning electron microscopy and transmission electron microscopy indicated that SPA significantly affected the morphology and ultrastructure of D. mahothocarpus hyphae, revealing that SPA can destroy the mycelial morphology and cell structure, especially the cell membrane of D. mahothocarpus. Furthermore, transcriptome analysis revealed that SPA significantly suppressed the expression of genes involved in morphology, cell membrane permeability, and oxidative stress. Then, we also found that SPA significantly promoted the accumulation of reactive oxygen species (ROS) in of D. mahothocarpus, while it decreased the content of reduced glutathione, inhibited the enzyme activities of superoxide dismutase and catalase, and exacerbated DNA damage. Annexin V-FITC/PI staining also confirmed that 1.0 mg/mL SPA could significantly induce apoptosis and necrosis. Discussion: Generally, SPA can induce ROS-mediated oxidative stress and cell death, thus destroying the cell membrane and hyphal morphology, and ultimately inhibiting mycelial growth, which indicates that SPA has multiple modes of action, providing a scientific basis for the use of SPA as an alternative plant-derived photoactivated fungicide against C. oleifera leaf spot blight. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2- induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugarspecific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. zooepidemicus (SEZ) is a significant zoonotic pathogen that causes septicemia, meningitis, and mastitis in domestic animals. Recent reports have highlighted high-mortality outbreaks among swine in the United States. Traditionally recognized for its adaptive immune functions, the CRISPR-Cas system has also been implicated in gene regulation, bacterial pathophysiology, virulence, and evolution. The Type I-C CRISPR-Cas system, which is prevalent in SEZ isolates, appears to play a pivotal role in regulating the pathogenicity of SEZ. By constructing a Cas3 mutant strain (ΔCas3) and a CRISPR-deficient strain (ΔCRISPR), we demonstrated that this system significantly promotes biofilm formation and cell adhesion. However, the deficiency in the CRISPR-Cas system did not affect bacterial morphology or capsule production. In vitro studies showed that the CRISPR-Cas system enhances pro-inflammatory responses in RAW264.7 cells. The ΔCas3 and ΔCRISPR mutant strains exhibited reduced mortality rates in mice, accompanied by a decreased bacterial load in specific organs. RNA-seq analysis revealed distinct expression patterns in both mutant strains, with ΔCas3 displaying a broader range of differentially expressed genes, which accounted for over 70% of the differential genes observed in ΔCRISPR. These genes were predominantly linked to lipid metabolism, the ABC transport system, signal transduction, and quorum sensing. These findings enhance our understanding of the complex role of the CRISPR-Cas system in SEZ pathogenesis and provide valuable insights for developing innovative therapeutic strategies to combat infections. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathologicalmanifestations in the pancreas after intraperitoneal infection. Long- time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis. [ABSTRACT FROM AUTHOR]

Bioinformatic studies on small proteins are under-represented due to difficulties in annotation posed by their small size. However, recent discoveries emphasize the functional significance of small proteins in cellular processes including cell signaling, metabolism, and adaptation to stress. In this study, we utilized a Random Forest classifier trained on sequence features, RNA-Seq, and Ribo- Seq data to uncover small proteins (smORFs) in M. tuberculosis. Independent predictions for the exponential and starvation conditions resulted in 695 potential smORFs. We examined the functional implications of these smORFs using homology searches, LC-MS/MS, and ChlP-seq data, testing their expression in diverse growth conditions, and identifying protein domains. We provide evidence that some of these smORFs could be part of operons, or exist as upstream ORFs. This expanded data resource for the proteins of M. tuberculosis would aid in fine-tuning the existing protein and gene regulatory networks, thereby improving system-wide studies. The primary goal of this study was to uncover and characterize smORFs in M. tuberculosis through bioinformatic analysis, shedding light on their functional roles and genomic organization. Further investigation of these potential smORFs would provide valuable insights into the genome organization and functional diversity of the M. tuberculosis proteome. [ABSTRACT FROM AUTHOR]

Pine wilt disease caused by Bursaphelenchus xylophilus poses a serious threat to the economic and ecological value of forestry. Nematode trapping fungi trap and kill nematodes using specialized trapping devices, which are highly efficient and non-toxic to the environment, and are very promising for use as biological control agents. In this study, we isolated several nematode-trapping fungi from various regions and screened three for their high nematocidal efficiency. However, the effectiveness of these fungi as nematicides is notably influenced by temperature and exhibits different morphologies in response to temperature fluctuations, which are categorized as "NA," "thin," "dense," and "sparse." The trend of trap formation with temperature was consistent with the trend of nematocidal efficiency with temperature. Both of which initially increased and then decreased with increasing temperature. Among them, Arthrobotrys cladodes exhibited the highest level of nematocidal activity and trap formation among the tested species. Transcriptome data were collected from A. cladodes with various trap morphologies. Hydrolase activity was significantly enriched according to GO and KEGG enrichment analyses. Eight genes related to hydrolases were found to be consistent with the trend of trap morphology with temperature. Weighted gene co-expression analysis and the Cytoscape network revealed that these 8 genes are associated with either mitosis or autophagy. This suggests that they contribute to the formation of "dense" structures in nematode-trapping fungi. One of these genes is the serine protein hydrolase gene involved in autophagy. This study reveals a potentially critical role for hydrolases in trap formation and nematocidal efficiency. And presents a model where temperature affects trap formation and nematocidal efficiency by influencing the serine protease prb1 involved in the autophagy process. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state. [ABSTRACT FROM AUTHOR]

Astaxanthin has multiple physiological functions and is applied widely. The yeast Phaffia rhodozyma is an ideal source of microbial astaxanthin. However, the stress conditions beneficial for astaxanthin synthesis often inhibit cell growth, leading to low productivity of astaxanthin in this yeast. In this study, 1 mg/L melatonin (MT) could increase the biomass, astaxanthin content, and yield in P. rhodozyma by 21.9, 93.9, and 139.1%, reaching 6.9 g/L, 0.3 mg/g DCW, and 2.2 mg/L, respectively. An RNA-seq-based transcriptomic analysis showed that MT could disturb the transcriptomic profile of P. rhodozyma cell. Furthermore, differentially expressed gene (DEG) analysis show that the genes induced or inhibited significantly by MT were mainly involved in astaxanthin synthesis, metabolite metabolism, substrate transportation, anti-stress, signal transduction, and transcription factor. A mechanism of MT regulating astaxanthin synthesis was proposed in this study. The mechanism is that MT entering the cell interacts with components of various signaling pathways or directly regulates their transcription levels. The altered signals are then transmitted to the transcription factors, which can regulate the expressions of a series of downstream genes as the DEGs. A zinc finger transcription factor gene (ZFTF), one of the most upregulated DEGs, induced by MT was selected to be overexpressed in P. rhodozyma. It was found that the biomass and astaxanthin synthesis of the transformant were further increased compared with those in MT-treatment condition. Combining MT-treatment and ZFTF overexpression in P. rhodozyma, the biomass, astaxanthin content, and yield were 8.6 g/L, 0.6 mg/g DCW, and 4.8 mg/L and increased by 52.1, 233.3, and 399.7% than those in the WT strain under MT-free condition. In this study, the synthesis and regulation theory of astaxanthin is deepened, and an efficient dual strategy for industrial production of microbial astaxanthin is proposed. [ABSTRACT FROM AUTHOR]

Introduction: Arbuscular mycorrhizal fungi (AMF) have been demonstrated their ability to enhance the arsenic (As) tolerance of host plants, and making the utilization of mycorrhizal plants a promising and practical approach for remediating As-contaminated soils. However, comprehensive transcriptome analysis to reveal the molecular mechanism of As tolerance in the symbiotic process between AMF and host plants is still limited. Methods: In this study, transcriptomic analysis of Gossypium seedlings was conducted with four treatments: non-inoculated Gossypium under non-As stress (CK0), non-inoculated Gossypium under As stress (CK100), F. mosseaeinoculated Gossypium under non-As stress (FM0), and F. mosseae-inoculated Gossypium under As stress (FM100). Results: Our results showed that inoculation with F. mosseae led to a reduction in net fluxes of Ca2+, while increasing Ca2+ contents in the roots and leaves of Gossypium under the same As level in soil. Notably, 199 and 3129 differentially expressed genes (DEGs) were specially regulated by F. mosseae inoculation under As stress and non-As stress, respectively. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analyses, we found that under As stress, F. mosseae inoculation up-regulated a significant number of genes related to the Ca2+ signaling pathway genes, involved in cellular process, membrane part, and signal transduction. This suggests a potential role in mitigating As tolerance in Gossypium seedlings. Furthermore, our analysis identified specific DEGs in transcription factor families, including ERF, MYB, NAC, and WRKY, that were upregulated by F. mosseae inoculation. Conversely, MYB and HB-other were down-regulated. The ERF and MYB families exhibited the highest number of up- and down-regulated DEGs, respectively, which were speculated to play an important role in alleviating the As toxicity of Gossypium. Discussion: Our findings provided valuable insights into the molecular theoretical basis of the Ca2+ signaling pathway in improving As tolerance of mycorrhizal plants in the future. [ABSTRACT FROM AUTHOR]

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation. [ABSTRACT FROM AUTHOR]

Introduction: Potatoes (Solanum tuberosum L.) can be infected by various viruses, but out of all of viruses, the potato virus Y (PVY) is the most detrimental. Research shows that the potato cultivar YouJin is especially vulnerable to PVY and displays severe symptoms, including leaf vein chlorosis, curled leaf margins, large necrotic spots on the leaf blades, and the growth of small new leaves. Methods: PVY infection in potato cultivar YouJin was confirmed through symptom observation, RT-PCR, and Western blot analysis. Transcriptome sequencing was used to analyze the genes associated with PVY pathogenesis in this cultivar. Result: Transcriptome analysis of differential genes was conducted in this study to examine the pathogenesis of PVY on YouJin. The results showed that 1,949 genes were differentially regulated, including 853 upregulated genes and 1,096 downregulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that carbohydrate synthesis and metabolism pathways were suppressed, and electron transferase and hydrolase activities were reduced. Moreover, there were increased expression levels of protein kinase genes. By focusing on plant–pathogen interaction pathways, six core genes all upregulating the WARK family of transcription factors were obtained. Additionally, a constructed PPI network revealed the identification of key modular differential genes, such as downregulated photosynthesis-related protein genes and upregulated AP2/ ERF-ERF transcription factors. Functional network enrichment analysis revealed that PVY infection limited RNA metabolism, glutathionylation, and peroxiredoxin activity while triggering the expression of associated defense genes in YouJin. After analyzing the above, 26 DEGs were screened and 12 DEGs were confirmed via RT-qPCR. Conclusion: These results establish a hypothetical framework for clarifying the pathogenesis of PVY in the YouJin variety of potatoes, which will help design the disease resistance of YouJin. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common foodborne pathogen which is frequently used as the reference strain for Salmonella. Investigating the sigma factor network and protomers is crucial to understand the genomic and transcriptomic properties of the bacterium. Its promoters were identified using various methods such as dRNA-seq, ChIP-chip, or ChIP-Seq. However, validation using ChIP-exo, which exhibits higher-resolution performance compared to conventional ChIP, has not been conducted to date. In this study, using the representative strain S. Typhimurium LT2 (LT2), the ChIP-exo experiment was conducted to accurately determine the binding sites of catalytic RNA polymerase subunit RpoB and major sigma factors (RpoD, RpoN, RpoS, and RpoE) during exponential phase. Integrated with the results of RNA-Seq, promoters and sigmulons for the sigma factors and their association with RpoB have been discovered. Notably, the overlapping regions among binding sites of each alternative sigma factor were found. Furthermore, comparative analysis with Escherichia coli str. K-12 substr. MG1655 (MG1655) revealed conserved binding sites of RpoD and RpoN across different species. In the case of small RNAs (sRNAs), 50 sRNAs observed their expression during the exponential growth of LT2. Collectively, the integration of ChIP-exo and RNA-Seq enables genome-scale promoter mapping with high resolution and facilitates the characterization of binding events of alternative sigma factors, enabling a comprehensive understanding of the bacterial sigma factor network and condition-specific active promoters. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

Bioinformatic studies on small proteins are under-represented due to difficulties in annotation posed by their small size. However, recent discoveries emphasize the functional significance of small proteins in cellular processes including cell signaling, metabolism, and adaptation to stress. In this study, we utilized a Random Forest classifier trained on sequence features, RNA-Seq, and Ribo-Seq data to uncover small proteins (smORFs) in M. tuberculosis. Independent predictions for the exponential and starvation conditions resulted in 695 potential smORFs. We examined the functional implications of these smORFs using homology searches, LC-MS/MS, and ChIP-seq data, testing their expression in diverse growth conditions, and identifying protein domains. We provide evidence that some of these smORFs could be part of operons, or exist as upstream ORFs. This expanded data resource for the proteins of M. tuberculosis would aid in fine-tuning the existing protein and gene regulatory networks, thereby improving system-wide studies. The primary goal of this study was to uncover and characterize smORFs in M. tuberculosis through bioinformatic analysis, shedding light on their functional roles and genomic organization. Further investigation of these potential smORFs would provide valuable insights into the genome organization and functional diversity of the M. tuberculosis proteome.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

This document is a corrigendum for an article titled "Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state." The corrigendum acknowledges errors in the legend and figure of the original article. The authors apologize for the errors and state that they do not affect the scientific conclusions of the article. The corrected figure and its caption are provided in the corrigendum. [Extracted from the article]

IntroductionArbuscular mycorrhizal fungi (AMF) have been demonstrated their ability to enhance the arsenic (As) tolerance of host plants, and making the utilization of mycorrhizal plants a promising and practical approach for remediating As-contaminated soils. However, comprehensive transcriptome analysis to reveal the molecular mechanism of As tolerance in the symbiotic process between AMF and host plants is still limited.MethodsIn this study, transcriptomic analysis of Gossypium seedlings was conducted with four treatments: non-inoculated Gossypium under non-As stress (CK0), non-inoculated Gossypium under As stress (CK100), F. mosseae-inoculated Gossypium under non-As stress (FM0), and F. mosseae-inoculated Gossypium under As stress (FM100).ResultsOur results showed that inoculation with F. mosseae led to a reduction in net fluxes of Ca2+, while increasing Ca2+ contents in the roots and leaves of Gossypium under the same As level in soil. Notably, 199 and 3129 differentially expressed genes (DEGs) were specially regulated by F. mosseae inoculation under As stress and non-As stress, respectively. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and enrichment analyses, we found that under As stress, F. mosseae inoculation up-regulated a significant number of genes related to the Ca2+ signaling pathway genes, involved in cellular process, membrane part, and signal transduction. This suggests a potential role in mitigating As tolerance in Gossypium seedlings. Furthermore, our analysis identified specific DEGs in transcription factor families, including ERF, MYB, NAC, and WRKY, that were upregulated by F. mosseae inoculation. Conversely, MYB and HB-other were down-regulated. The ERF and MYB families exhibited the highest number of up- and down-regulated DEGs, respectively, which were speculated to play an important role in alleviating the As toxicity of Gossypium.DiscussionOur findings provided valuable insights into the molecular theoretical basis of the Ca2+ signaling pathway in improving As tolerance of mycorrhizal plants in the future.

Bordetella bronchiseptica is a highly contagious respiratory bacterial veterinary pathogen. In this study the contribution of the transcriptional regulators BvgR, RisA, RisS, and the phosphorylation of RisA to global gene regulation, intracellular cyclic-di-GMP levels, motility, and biofilm formation were evaluated. Next Generation Sequencing (RNASeq) was used to differentiate the global gene regulation of both virulence-activated and virulence-repressed genes by each of these factors. The BvgAS system, along with BvgR, RisA, and the phosphorylation of RisA served in cyclic-di-GMP degradation. BvgR and unphosphorylated RisA were found to temporally regulate motility. Additionally, BvgR, RisA, and RisS were found to be required for biofilm formation.

Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

Brown film formation, a unique developmental stage in the life cycle of Lentinula edodes, is essential for the subsequent development of fruiting bodies in L. edodes cultivation. The pH of mushroom growth substrates are usually adjusted with hydrated lime, yet the effects of hydrated lime on cultivating L. edodes and the molecular mechanisms associated with the effects have not been studied systemically. We cultivated L. edodes on substrates supplemented with 0% (CK), 1% (T1), 3% (T2), and 5% (T3) hydrated lime (Ca (OH)2), and applied transcriptomics and qRT-PCR to study gene expression on the brown film formation stage. Hydrated lime increased polysaccharide contents in L. edodes, especially in T2, where the 5.3% polysaccharide content was approximately 1.5 times higher than in the CK. The addition of hydrated lime in the substrate promoted laccase, lignin peroxidase and manganese peroxidase activities, implying that hydrated lime improved the ability of L. edodes to decompose lignin and provide nutrition for its growth and development. Among the annotated 9,913 genes, compared to the control, 47 genes were up-regulated and 52 genes down-regulated in T1; 73 genes were up-regulated and 44 were down-regulated in T2; and 125 genes were up-regulated and 65 genes were down-regulated in T3. Differentially expressed genes (DEGs) were enriched in the amino acid metabolism, lipid metabolism and carbohydrate metabolism related pathways. The carbohydrate-active enzyme genes up-regulated in the hydrated lime treatments were mostly glycosyl hydrolase genes. The results will facilitate future optimization of L. edodes cultivation techniques and possibly shortening the production cycle. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are important for plant defense against fungal attack. As a necrotrophic fungus, Botrytis cinerea can exploit ROS that originated from both sides of the host and pathogen during interaction to facilitate its infestation. Meanwhile, B. cinerea needs to exert an efficient oxidative stress responsive system to balance the intracellular redox state when encountering deleterious ROS levels. However, the machinery applied by B. cinerea to cope with ROS remains obscure. Herein, we investigated the role of the transcription factor BcMsn2 in regulating B. cinerea redox homeostasis. Disruption of the BcMsn2 gene severely impaired vegetative growth, sclerotium formation, conidial yield, and fungal virulence. The intracellular oxidative homeostasis of the Δbcmsn2 mutant was disrupted, leading to significantly elevated levels of ROS and reduced activities of enzymes closely associated with oxygen stress, such as catalase (CAT) and superoxide dismutase (SOD). RNA-Seq and qRT-PCR analyses showed remarkable downregulation of the expression of several genes encoding ROS scavenging factors involved in maintaining the redox homeostasis in Δbcmsn2, suggesting that BcMsn2 functions as a transcriptional regulator of these genes. Our findings indicated that BcMsn2 plays an indispensable role in maintaining the equilibrium of the redox state in B. cinerea, and intracellular ROS serve as signaling molecules that regulate the growth, asexual reproduction, and virulence of this pathogen. [ABSTRACT FROM AUTHOR]

Emerging multidrug resistance (MDR) in Clarireedia spp. is a huge challenge to the management of dollar spot (DS) disease on turfgrass. Insight into the molecular basis of resistance mechanisms may help identify key molecular targets for developing novel effective chemicals. Previously, a MDR isolate (LT586) of C. jacksonii with significantly reduced sensitivities to propiconazole, boscalid, and iprodione, and a fungicide-sensitive isolate (LT15) of the same species were isolated from creeping bentgrass (Agrostis stolonifera L.). The present study aimed to further explore the molecular mechanisms of resistance by using genome-wide transcriptional analyses of the two isolates. A total of 619 and 475 differentially expressed genes (DEGs) were significantly down and upregulated in the MDR isolate LT586, compared with the sensitive isolate LT15 without fungicide treatment. Three hundreds and six and 153 DEGs showed significantly lower and higher expression in the MDR isolate LT586 than those in the sensitive isolate LT15, which were commonly induced by the three fungicides. Most of the 153 upregulated DEGs were xenobiotic detoxification-related genes and genes with transcriptional functions. Fifty and 17 upregulated DEGs were also commonly observed in HRI11 (a MDR isolate of the C. jacksonii) compared with the HRS10 (a fungicide-sensitive isolate of same species) from a previous study without and with the treatment of propiconazole, respectively. The reliability of RNA-seq data was further verified by qRT-PCR method using a few select potentially MDR-related genes. Results of this study indicated that there were multiple uncharacterized genes, possibly responsible for MDR phenotypes in Clarireedia spp., which may have important implications in understanding the molecular mechanisms underlying MDR resistance. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus pentosus (Lbp. pentosus) is a species of lactic acid bacteria with a great relevance during the table olive fermentation process, with ability to form non-pathogenic biofilms on olive epidermis. The objective of this work is to deepen into the genetic mechanisms of adaptation of Lpb. pentosus LPG1 during Spanish-style green table olive fermentations, as well as to obtain a better understanding of the mechanisms of adherence of this species to the fruit surface. For this purpose, we have carried out a transcriptomic analysis of the differential gene expression of this bacterium during 60 days of fermentation in both brine and biofilms ecosystems. In brines, it was noticed that a total of 235 genes from Lpb. pentosus LPG1 were differentially expressed during course of fermentation and grouped into 9 clusters according to time-course analysis. Transport and metabolism of carbohydrates and amino acids, energy production, lactic acid and exopolysaccharide synthesis genes increased their expression in the planktonic cells during course of fermentation. On the other hand, expression of genes associated to stress response, bacteriocin synthesis and membrane protein decreased. A total of 127 genes showed significant differential expression between Lpb. pentosus LPG1 planktonic (brine) and sessile (biofilms) cells at the end of fermentation process (60 days). Among the 64 upregulated genes in biofilms, we found genes involved in adhesion (strA), exopolysaccharide production (ywqD, ywqE, and wbnH), cell shape and elongation (MreB), and well as prophage excision. Deeping into the genetic bases of beneficial biofilm formation by Lpb. pentosus strains with probiotic potential will help to turn this fermented vegetable into a carrier of beneficial microorganisms to the final consumers. [ABSTRACT FROM AUTHOR]

Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli. [ABSTRACT FROM AUTHOR]

Introduction: Invasive fungal infections (IFIs) are fatally threatening to critical patients. The fungal defensin as an antifungal protein can widely inhibit fungi. Methods: In this study, eight antifungal genes from different filamentous fungi were optimized by synonymous codon bias and heterologously expressed in Escherichia coli. Results and discussion: Only the antifungal protein (AFP) from Aspergillus giganteus was produced, whereas the AFP from its mutation of the chitin-binding domain could not be expressed, thereby suggesting the importance of the motif for protein folding. In addition, the recombinant AFP (rAFP, 100 µg/mL) pre-heated at 50 °C for 1 h effectively inhibited Paecilomyces variotii CICC40716 of IFIs by 55%, and no cell cytotoxicity was observed in RAW264.7 cells. After being pre- heated at 50C for 8 h, the fluorescence emission intensity of the rAFP decreased and shifted from 343 nm to 335 nm. Moreover, the helix and b-turn of the rAFP gradually decreased with the pre-heated treatment temperature of 50C via circular dichroism spectroscopy. Propidium iodide staining revealed that the rAFP could cause damage to the cell membrane. Moreover, the corresponding differentially expressed genes (DEGs) for downregulation such as amino sugar and nucleotide sugar metabolism, as well as mitogen-activated protein kinase (MAPK) signaling pathway involved in the cell wall integrity were found via the RNA-seq of rAFP treatment. By contrast, the upregulated DEGs were enriched in response to the oxidative stress of Biological Process by the Gene Ontology (GO) database. The encoding proteins of laccase, multicopper oxidase, and nitroreductase that contributed to reactive oxygen species (ROS) scavenging could be recognized. These results suggested that the rAFP may affect the integrity of the cell wall and cell membrane, and promote the increase in ROS, thereby resulting in fungal death. Consequently, drug development could be based on the inhibitory effect of the rAFP on IFIs. [ABSTRACT FROM AUTHOR]

Introduction: Candida auris is a newly emerging pathogenic fungus of global concern and has been defined by the World Health Organization (WHO) as a member of the critical group of the most health-threatening fungi. Methods: This study reveals and reports for the first time that a rough morphotype C. auris strain causes urinary tract infections in non-intensive care unit (ICU) inpatients. Furthermore, the morphology, the scanning electronmicroscopy (SEM), Whole-genome resequencing and RNA sequencing of C. auris possessing rough morphotype colonies compared to their smooth morphotype counterparts. Results: The newly identified phenotypic variation of C. auris appears round, convex, dry, and burr-like with a rough texture. SEM shows that rough type C. auris has a rough and uneven colony surface with radial wrinkles and irregular spore arrangement. Cells of the rough morphotype C. auris naturally aggregate into clusters with tight connections in the liquid, and it seems that the cell division is incomplete. A genome-wide analysis of the rough type C. auris confirmed its genetic association with the smooth type of C. auris prevalent in China (Shenyang) two years ago; however, single nucleotide polymorphism (SNP) mutations of five genes (ACE2, IFF6, RER2, UTP20, and CaO19.5847) were identified more recently. RNA-seq revealed IFF2/HYR3, DAL5, PSA31, and SIT1 were notably up-regulated, while multiple cell wall-associated genes (ALS1, MNN1, PUL1, DSE1, SCW11, PGA38, RBE1, FGR41, BGLI, GIT3, CEP3, and SAP2) were consistently down-regulated in rough morphotype C. auris. Discussion: The rough phenotypic variation of C. auris is likely to be related to the structural and functional changes in cell wall proteins. This novel rough morphotype C. auris will provide a basis for further studies concerning the evolutionary characteristics of C. auris. [ABSTRACT FROM AUTHOR]

Pitaya canker, caused by Neoscytalidium dimidiatum, is one of the most important fungal diseases that cause significant losses in production. To replace chemical pesticides, the use of biocontrol strains to manage plant diseases has been the focus of research. In this study, the bacterial strain AF01, identified as Paenibacillus polymyxa, exhibited significant antifungal effects against N. dimidiatum and four other pitaya fungal pathogens. The strain P. polymyxa AF01 produces 13 fusaricidins, which directly inhibit mycelial growth, spore germination and germ tube elongation by causing the membrane integrity and cell ultrastructure to incur irreversible damage. Pot experiment and yield test confirmed that AF01 provided preservative effects by reducing the disease index. In comparison to the untreated control groups, RNA-seq data showed that P. polymyxa AF01 selectively blocked some transcription and translation processes and inhibited RNA and DNA structural dynamics, energy production and conversion, and signal transduction, particularly cell wall biosynthesis, changes in membrane permeability, and impairment of protein biosynthesis. Thus, P. polymyxa AF01 could be potentially useful as a suitable biocontrol agent for pitaya canker. [ABSTRACT FROM AUTHOR]

Introduction: Pseudorabies virus (PRV) is a herpesvirus that can infect domestic animals, such as pigs, cattle and sheep, and cause fever, itching (except pigs), and encephalomyelitis. In particular, the emergence of PRV variants in 2011 have resulted in serious economic losses to the Chinese pig industry. However, the signaling pathways mediated by PRV variants and their related mechanisms are not fully understood. Methods: Here, we performed RNA-seq to compare the gene expression profiling between PRV virulent SD2017-infected PK15 cells and Bartha-K/61-infected PK15 cells. Results: The results showed that 5,030 genes had significantly different expression levels, with 2,239 upregulated and 2,791 downregulated. GO enrichment analysis showed that SD2017 significantly up-regulated differentially expressed genes (DEGs) were mainly enriched in the binding of cell cycle, protein and chromatin, while down-regulated DEGs weremainly enriched in ribosomes. KEGG enrichment analysis revealed that the pathways most enriched for upregulated DEGs were pathways in cancer, cell cycle, microRNAs in cancer, mTOR signaling pathway and autophagy-animal. The most down-regulated pathways of DEGs enrichment were ribosome, oxidative phosphorylation, and thermogenesis. These KEGG pathways were involved in cell cycle, signal transduction, autophagy, and virus-host cell interactions. Discussion: Our study provides a general overview of host cell responses to PRV virulent infection and lays a foundation for further study of the infection mechanism of PRV variant strain. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus pentosus (Lbp. pentosus) is a species of lactic acid bacteria with a great relevance during the table olive fermentation process, with ability to form non-pathogenic biofilms on olive epidermis. The objective of this work is to deepen into the genetic mechanisms of adaptation of Lpb. pentosus LPG1 during Spanish-style green table olive fermentations, as well as to obtain a better understanding of the mechanisms of adherence of this species to the fruit surface. For this purpose, we have carried out a transcriptomic analysis of the differential gene expression of this bacterium during 60 days of fermentation in both brine and biofilms ecosystems. In brines, it was noticed that a total of 235 genes from Lpb. pentosus LPG1 were differentially expressed during course of fermentation and grouped into 9 clusters according to time-course analysis. Transport and metabolism of carbohydrates and amino acids, energy production, lactic acid and exopolysaccharide synthesis genes increased their expression in the planktonic cells during course of fermentation. On the other hand, expression of genes associated to stress response, bacteriocin synthesis and membrane protein decreased. A total of 127 genes showed significant differential expression between Lpb. pentosus LPG1 planktonic (brine) and sessile (biofilms) cells at the end of fermentation process (60 days). Among the 64 upregulated genes in biofilms, we found genes involved in adhesion (strA), exopolysaccharide production (ywqD, ywqE, and wbnH), cell shape and elongation (MreB), and well as prophage excision. Deeping into the genetic bases of beneficial biofilm formation by Lpb. pentosus strains with probiotic potential will help to turn this fermented vegetable into a carrier of beneficial microorganisms to the final consumers.

Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli.

Green mold caused by Trichoderma spp. has become one of the most serious diseases which threatening the production of Ganoderma lingzhi. To understand the possible resistance mechanism of the G. lingzhi response to T. hengshanicum infection, we examined the G. lingzhi transcript accumulation at 0, 12, and 24 h after T. hengshanicum inoculation. The gene expression analysis was conducted on the interaction between G. lingzhi and T. hengshanicum using RNA-seq and digital gene expression (DGE) profiling methods. Transcriptome sequencing indicated that there were 162 differentially expressed genes (DEGs) at three infection time points, containing 15 up-regulated DEGs and 147 down-regulated DEGs. Resistance-related genes thaumatin-like proteins (TLPs) (PR-5s), phenylalanine ammonia-lyase, and Beta-1,3-glucan binding protein were significantly up-regulated. At the three time points of infection, the heat shock proteins (HSPs) genes of G. lingzhi were down-regulated. The down-regulation of HSPs genes led to the inhibition of HSP function, which may compromise the HSP-mediated defense signaling transduction pathway, leading to G. lingzhi susceptibility. Pathway enrichment analyses showed that the main enriched pathways by G. lingzhi after infection were sphingolipid metabolism, ether lipid metabolism, and valine, leucine and isoleucine degradation pathway. Overall, the results described here improve fundamental knowledge of molecular responses to G. lingzhi defense and contribute to the design of strategies against Trichoderma spp. [ABSTRACT FROM AUTHOR]

Isochrysis galbana, as a potential accumulator of fucoxanthin, has become a valuable material to develop functional foods for humans. Our previous research revealed that green light effectively promotes the accumulation of fucoxanthin in I. galbana, but there is little research on chromatin accessibility in the process of transcriptional regulation. This study was conducted to reveal the mechanism of fucoxanthin biosynthesis in I. galbana under green light by analyzing promoter accessibility and gene expression profiles. Differentially accessible chromatin regions (DARs)- associated genes were enriched in carotenoid biosynthesis and photosynthesis-antenna protein formation, including IgLHCA1, IgLHCA4, IgPDS, IgZ-ISO, IglcyB, IgZEP, and IgVDE. The motifs for the MYB family were also identified as candidates controlling metabolic regulation responses to green light culture of I. galbana, including IgMYB1, IgMYB2, IgMYB33, IgMYB42, IgMYB98, IgMYB118, and IgMYB119. The results of differential expression analysis and WGCNA showed that several genes or transcription factors (TFs) related to carotenoid metabolism and photosynthesis exhibited a higher expression level and were significantly upregulated in A-G5d compared with A-0d and A-W5d, including IgMYB98, IgLHCA1, IgLHCX2, IgLHCB4, and IgLHCB5. This suggests that upregulation of these genes by green light may be the key factor leading to fucoxanthin accumulation by regulating the photosynthesis-antenna protein pathway. An integrated analysis of ATAC-seq and RNA-seq showed that 3 (IgphoA, IgPKN1, IgOTC) of 34 DARs-associated genes displayed obvious changes in their chromatin regions in ATAC-seq data, suggesting that these genes specific for green light may play a key role in fucoxanthin biosynthesis in I. galbana through a complex regulatory network of multiple metabolic pathways interacting with each other. These findings will facilitate in-depth understanding the molecular regulation mechanisms of fucoxanthin in I. galbana and its role in response to green light regulation, providing technical support for the construction of high fucoxanthin content strains. [ABSTRACT FROM AUTHOR]

Critically ill patients have higher risk of serious fungal infections, such as invasive aspergillosis (IA) which is mainly caused by the human fungal pathogen Aspergillus fumigatus. Triazole drugs are the primary therapeutic agents for the first-line treatment of IA, which could easily cause drug resistance problems. Here, we assess the potential of AgNPs synthesized with Artemisia argyi leaf extract and domiphen as new antifungal agents to produce synergistic antimicrobial effects on Aspergillus fumigatus, and dissect possible molecular mechanisms of action. Plate inoculation assays combined with drug susceptibility test and cytotoxicity test showed that the combination of AgNPs and domiphen has synergistic antimicrobial effects on A. fumigatus with low cytotoxicity. Gene Ontology (GO) enrichment analysis showed that AgNPs and domiphen inhibit the growth of A. fumigatus by suppressing nitrate assimilation, and purine nucleobase metabolic process and amino acid transmembrane transport, respectively. When the two drugs are combined, AgNPs has epistatic effects on domiphen. Moreover, the combination of AgNPs and domiphen primarily influence secondary metabolites biosynthesis, steroid biosynthesis and nucleotide sugar metabolism of A. fumigatus via Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, protein-protein interactions (PPI) analysis combined with validation experiments showed that the combination of AgNPs and domiphen could enhance the expression of copper transporter and inhibit nitrogen source metabolism. In addition, the synergistic antimicrobial effects could be enhanced or eliminated depending on exogenous addition of copper and nitrogen source, respectively. Taken together, the results of this study provide a theoretical basis and a new strategy for the treatment of IA. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNASeq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs), as key regulators, are closely associated with the development of a variety of disease. However, the mechanisms by which lncRNAs regulate Clostridium perfringens type C induced piglet diarrhea are unclear. Methods: In the present study, we explored the expression and characterization of lncRNAs in a C. perfringens beta2 (CPB2) toxin-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) using RNA-sequencing (RNA-seq). Results: A total of 6,558 lncRNAs were identified, of which 49 lncRNAs were significantly differentially expressed between the control and CPB2 groups. Functional enrichment analysis showed that the target genes of differentially expressed lncRNA EN-90756 were mainly associated with defense response to virus, and negative regulation of apoptotic process. LncRNA EN-90756 was significantly up-regulated in IPEC-J2 cells at different time points after CPB2 treatment. Functionally, knockdown of lncRNA EN-90756 might regulate the proliferation and apoptosis of IPEC-J2 cells by affecting the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. LncRNA EN-90756 may be involved in CPB2 toxin-induced piglet diarrhea by regulating the expression of its target gene MX1 (encoding MX dynamin like GTPase 1). Conclusion: Long non-coding RNA EN-90756 affected the antiviral ability of IPEC-J2 cells by regulating the expression of MX1. Meanwhile, lncRNA EN-90756 might regulate cell proliferation and apoptosis by affecting JAK-STAT signaling pathway activation. These findings provide novel perspectives and directions for further exploration of the regulatory mechanisms of lncRNAs on CPB2 toxin-induced diarrhea in piglets. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused a global outbreak of coronavirus disease 2019 (COVID-19) pandemic. To elucidate the mechanism of SARS-CoV-2 replication and immunogenicity, we performed a comparative transcriptome profile of mRNA and long non-coding RNAs (lncRNAs) in human lung epithelial cells infected with the SARS-CoV-2 wild-type strain (8X) and the variant with a 12-bp deletion in the E gene (F8). In total, 3,966 differentially expressed genes (DEGs) and 110 differentially expressed lncRNA (DE-lncRNA) candidates were identified. Of these, 94 DEGs and 32 DE-lncRNAs were found between samples infected with F8 and 8X. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzes revealed that pathways such as the TNF signaling pathway and viral protein interaction with cytokine and cytokine receptor were involved. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to the immune response, which might explain the different replication and immunogenicity properties of the 8X and F8 strains. These results provide a useful resource for studying the pathogenesis of SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Tardigrades, known colloquially as water bears or moss piglets, are diminutive animals capable of surviving many extreme environments, even been exposed to space in low Earth orbit. Recently termed tardigrade disordered proteins (TDPs) include three families as cytoplasmic-(CAHS), secreted- (SAHS), and mitochondrial-abundant heat soluble (MAHS) proteins. How these tiny animals survive these stresses has remained relatively mysterious. Cyanobacteria cast attention as a "microbial factory" to produce biofuels and high-value-added chemicals due to their ability to photosynthesis and CO2 sequestration. We explored a lot about biofuel stress and related mechanisms in Synechocystis sp. PCC 6803. The previous studies show that CAHS protein heterogenous expression in bacteria, yeast, and human cells increases desiccation tolerance in these hosts. In this study, the expression of three CAHS proteins in cyanobacterium was found to affect the tolerance to biofuels, while the tolerance to Cd2+ and Zn2+ were slightly affected in several mutants. A quantitative transcriptomics approach was applied to decipher response mechanisms at the transcriptional level further. [ABSTRACT FROM AUTHOR]

Objective: Bovine tuberculosis (bTB) is a chronic respiratory infectious disease of domestic livestock caused by intracellular Mycobacterium bovis infection, which causes ~$3 billion in annual losses to global agriculture. Providing novel tools for bTB managements requires a comprehensive understanding of the molecular regulatory mechanisms underlying the M. bovis infection. Nevertheless, a combination of different bioinformatics and systems biology methods was used in this study in order to clearly understand the molecular regulatory mechanisms of bTB, especially the immunomodulatory mechanisms of M. bovis infection. Methods: RNA-seq data were retrieved and processed from 78 (39 noninfected control vs. 39 M. bovis-infected samples) bovine alveolar macrophages (bAMs). Next, weighted gene co-expression network analysis (WGCNA) was performed to identify the co-expression modules in non-infected control bAMs as reference set. The WGCNA module preservation approach was then used to identify non-preserved modules between non-infected controls and M. bovis-infected samples (test set). Additionally, functional enrichment analysis was used to investigate the biological behavior of the non-preserved modules and to identify bTB-specific non-preserved modules. Co-expressed hub genes were identified based on module membership (MM) criteria of WGCNA in the non-preserved modules and then integrated with protein-protein interaction (PPI) networks to identify co-expressed hub genes/transcription factors (TFs) with the highest maximal clique centrality (MCC) score (hub-central genes). Results: As result, WGCNA analysis led to the identification of 21 modules in the non-infected control bAMs (reference set), among which the topological properties of 14 modules were altered in the M. bovis-infected bAMs (test set). Interestingly, 7 of the 14 non-preserved modules were directly related to the molecular mechanisms underlying the host immune response, immunosuppressive mechanisms of M. bovis, and bTB development. Moreover, among the co-expressed hub genes and TFs of the bTB-specific non-preserved modules, 260 genes/TFs had double centrality in both co-expression and PPI networks and played a crucial role in bAMs-M. bovis interactions. Some of these hub-central genes/TFs, including PSMC4, SRC, BCL2L1, VPS11, MDM2, IRF1, CDKN1A, NLRP3, TLR2, MMP9, ZAP70, LCK, TNF, CCL4, MMP1, CTLA4, ITK, IL6, IL1A, IL1B, CCL20, CD3E, NFKB1, EDN1, STAT1, TIMP1, PTGS2, TNFAIP3, BIRC3, MAPK8, VEGFA, VPS18, ICAM1, TBK1, CTSS, IL10, ACAA1, VPS33B, and HIF1A, had potential targets for inducing immunomodulatory mechanisms by M. bovis to evade the host defense response. Conclusion: The present study provides an in-depth insight into the molecular regulatory mechanisms behind M. bovis infection through biological investigation of the candidate non-preserved modules directly related to bTB development. Furthermore, several hub-central genes/TFs were identified that were significant in determining the fate of M. bovis infection and could be promising targets for developing novel anti-bTB therapies and diagnosis strategies. [ABSTRACT FROM AUTHOR]

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC–MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pulldown mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus. [ABSTRACT FROM AUTHOR]

Climate change affects animal physiology. In particular, rising ambient temperatures reduce animal vitality due to heat stress and this can be observed at various levels which included genome, transcriptome, and microbiome. In a previous study, microbiota highly associated with changes in cattle physiology, which included rectal temperature, drooling score and respiratory score, were identified under heat stress conditions. In the present study, genes differentially expressed between individuals were selected representing different additive genetic effects toward the heat stress response in cattle in their production condition. Moreover, a correlation network analysis was performed to identify interactions between the transcriptome and microbiome for 71 Chinese Holstein cows sequenced for mRNA from blood samples and for 16S rRNA genes from fecal samples. Bioinformatics analysis was performed comprising: i) clustering and classification of 16S rRNA sequence reads, ii) mapping cows' transcripts to the reference genome and their expression quantification, and iii) statistical analysis of both data types--including differential gene expression analysis and gene set enrichment analysis. A weighted co-expression network analysis was carried out to assess changes in the association between gene expression and microbiota abundance as well as to find hub genes/microbiota responsible for the regulation of gene expression under heat stress. Results showed 1,851 differentially expressed genes were found that were shared by three heat stress phenotypes. These genes were predominantly associated with the cytokine-cytokine receptor interaction pathway. The interaction analysis revealed three modules of genes and microbiota associated with rectal temperature with which two hubs of those modules were bacterial species, demonstrating the importance of the microbiome in the regulation of gene expression during heat stress. Genes and microbiota from the significant modules can be used as biomarkers of heat stress in cattle. [ABSTRACT FROM AUTHOR]

Solar crystallizer ponds are characterized by high population density with a relatively simple community structure in terms of species composition. The microbial community in the solar saltern of Santa Pola (Alicante, Spain), is largely dominated by the hyperhalophilic square archaeon Haloquadratum walsbyi. Here we studied metatranscriptomes retrieved from a crystallizer pond during the winter of 2012 and summer of 2014 and compared Hqr. walsbyi's transcription patterns with that of the cultured strain Hqr. walsbyi HBSQ001. Significant differences were found between natural and the cultured grown strain in the distribution of transcript levels per gene. This likely reflects the adaptation of the cultured strain to the relative homogeneous growth conditions while the natural species, which is represented by multiple ecotypes, is adapted to heterogeneous environmental conditions and challenges of nutrient competition, viral attack, and other stressors. An important consequence of this study is that expression patterns obtained under artificial cultivation conditions cannot be directly extrapolated to gene expression under natural conditions. Moreover, we found 195 significantly differential expressed genes between the seasons, with 140 genes being higher expressed in winter and mainly encode proteins involved in energy and carbon source acquiring processes, and in stress responses. [ABSTRACT FROM AUTHOR]

Stigma maydis polysaccharide (SMPS) is a plant polysaccharide that participates in immune regulation and gastrointestinal motility. Autism spectrum disorder (ASD) refers to a group of neurodevelopmental disorders, and ASD patients often present intestinal microflora imbalance problems; however, there is no effective treatment method. This study explores the effect of SMPS intervention on the gut microbiota in autism model rats as well as the potential action pathways. Female Wistar rats were intraperitoneally injected with sodium valproic acid (VPA) or normal saline at embryonic day 12.5 to establish an autism model or normal control in their offspring. The offspring prenatally exposed to VPA were randomly assigned to the VPA and the SMPS groups. The SMPS group was administered SMPS from E0.5 to postnatal day (PND) 21. We performed 16S rRNA and transcriptomics analyses to reveal the gut microbiota (GM) and differentially expressed genes in the autism model rats in response to SMPS intervention. SMPS intervention significantly improved the diversity and structure of the GM in autism model rats compared with the VPA rats. Moreover, the relative abundance of Prevotellaceae and Lachnospiraceae_NK4A136_group was increased after SMPS intervention. Transcriptome sequencing showed that 496 differentially expressed genes (DEGs) were identified after SMPS administration compared with the VPA group. Meanwhile, gene ontology (GO) enrichment analysis of DEGs was showed that the SMPS group had significant 653 GO terms. SMPS intervention had a major influence on oxidative phosphorylation, retrograde endocannabinoid signaling, thermogenesis, ribosome, protein digestion and absorption, renin-angiotensin system, calcium signaling pathway, glycosphingolipid biosynthesis-ganglio series, and propanoate metabolism pathways. Overall, this study suggests that SMPS interventions in early life may have an impact on gut microbiota, and then affect the transcriptomics levels of the hippocampal tissue in the VPA-induced autism model rats. It provides scientific evidence for the role of the microbe-gut-brain axis in ASD research. [ABSTRACT FROM AUTHOR]

While metagenome sequencing may provide insights on the genome sequences and composition of microbial communities, metatranscriptome analysis can be useful for studying the functional activity of a microbiome. RNA-Seq data provides the possibility to determine active genes in the community and how their expression levels depend on external conditions. Although the field of metatranscriptomics is relatively young, the number of projects related to metatranscriptome analysis increases every year and the scope of its applications expands. However, there are several problems that complicate metatranscriptome analysis: complexity of microbial communities, wide dynamic range of transcriptome expression and importantly, the lack of high-quality computational methods for assembling meta-RNA sequencing data. These factors deteriorate the contiguity and completeness of metatranscriptome assemblies, therefore affecting further downstream analysis. Here we present MetaGT, a pipeline for de novo assembly of metatranscriptomes, which is based on the idea of combining both metatranscriptomic and metagenomic data sequenced from the same sample. MetaGT assembles metatranscriptomic contigs and fills in missing regions based on their alignments to metagenome assembly. This approach allows to overcome described complexities and obtain complete RNA sequences, and additionally estimate their abundances. Using various publicly available real and simulated datasets, we demonstrate that MetaGT yields significant improvement in coverage and completeness of metatranscriptome assemblies compared to existing methods that do not exploit metagenomic data. The pipeline is implemented in NextFlow and is freely available from https://github.com/ablab/metaGT. [ABSTRACT FROM AUTHOR]

Cotton leaf curl Multan virus (CLCuMuV) is a whitefly-vectored begomovirus that poses ramping threat to several economically important crops worldwide. The differential transmission of CLCuMuV by its vector Bemisia tabaci mainly relies on the type of whitefly cryptic species. However, the molecular responses among different whitefly cryptic species in response to early CLCuMuV infection remain elusive. Here, we compared early-stage transcriptomic profiles of Asia II 7 and MEAM1 cryptic species infected by CLCuMuV. Results of Illumina sequencing revealed that after 6 and 12 h of CLCuMuV acquisition, 153 and 141 genes among viruliferous (VF) Asia II 7, while 445 and 347 genes among VF MEAM 1 whiteflies were differentially expressed compared with aviruliferous (AVF) whiteflies. The most abundant groups of differentially expressed genes (DEGs) among Asia II 7 and MEAM1 were associated with HTH-1 and zf-C2H2 classes of transcription factors (TFs), respectively. Notably, in contrast to Asia II 7, MEAM1 cryptic species displayed higher transcriptional variations with elevated immune-related responses following CLCuMuV infection. Among both cryptic species, we identified several highly responsive candidate DEGs associated with antiviral innate immunity (alpha glucosidase, LSM14-like protein B and phosphoenolpyruvate carboxykinase), lysosome (GPIanchored protein 58) and autophagy/phagosome pathways (sequestosome-1, cathepsin F-like protease), spliceosome (heat shock protein 70), detoxification (cytochrome P450 4C1), cGMP-PKG signaling pathway (myosin heavy chain), carbohydrate metabolism (alpha-glucosidase), biological transport (mitochondrial phosphate carrier) and protein absorption and digestion (cuticle protein 8). Further validation of RNA-seq results showed that 23 of 28 selected genes exhibited concordant expression both in RT-qPCR and RNA-seq. Our findings provide vital mechanistic insights into begomoviruswhitefly interactions to understand the dynamics of differential begomovirus transmission by different whitefly cryptic species and reveal novel molecular targets for sustainable management of insect-transmitted plant viruses. [ABSTRACT FROM AUTHOR]