ABSTRACT: Transcriptional profile of Streptococcus pyogenes stk mutant strain JRS2516 vs its wild type parent strain MGAS2221 The RNA prepared from triplicate cultures for the wild type and the stk mutant strains was each labeled with Cy3 and hybridized to duplicate arrays. Reference RNA consisted of pooled RNA for the wild type and stk mutant, labeled with Cy5.
Project description:Transcriptional profile of Streptococcus pyogenes stk mutant strain JRS2516 vs its wild type parent strain MGAS2221 Overall design: The RNA prepared from triplicate cultures for the wild type and the stk mutant strains was each labeled with Cy3 and hybridized to duplicate arrays. Reference RNA consisted of pooled RNA for the wild type and stk mutant, labeled with Cy5.
Project description:Genes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). We report that in GAS, stk is required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of ?-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that the stk deletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.
Project description:Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the ?stk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the ?stk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in ?stk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the ?stk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains.
Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from the SEEDSTICK mutant in Arabidopsis with a wild type, to unveil the role of this transcription factor in seed development and decipher the impact of this factor in PAs metabolism Methods: Total RNA was extracted from two biological replicates from both wild-type and stk mutant inflorescences and siliques until 5 DAP with the Qiagen Kit according to the manufacturer's instructions. DNA contaminations were removed using the PROMEGA RQ1 RNase-Free DNase according to the manufacturer's instructions. RNA quality integrity was analyzed by electrophoresis gel and was validated on a Bioanalyzer 2100 (Aligent, Santa Clara, CA); RNA Integrity Number (RIN) values were greater than 7 for all the samples. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5’-TGCGATGCAGAAGTTGCGCTC-3’) and RT 781 (5’-AGTACGCGGCATTGATTTCTTG-3’). Sequencing libraries were prepared according to the manufacturer’s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. The processing of fluorescent images into sequences, base-calling and quality value calculations were performed using the Illumina data processing pipeline (version 1.8). Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30% bases with Q < 20. Finally, a quality control of the raw sequence data was performed using FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/]. Results: A total of 102,278,242 reads passed a quality filter and 85% were mapped back to the Arabidopsis TAIR10 genome. Approximately 90% mapped uniquely to only one location and could be assigned to a single annotated TAIR10 gene. Normalization of expression values was performed using RPKM values. All other parameters were kept at default levels. The CLC Genomic Workbench was also further used to determine all differentially expressed transcripts found in each cDNA library. Baggerley's test and a FDR correction were used for statistical analysis of samples. Our analysis revealed that 156 genes were upregulated, whereas for 90 genes a reduction in their mRNA level was observed in the stk mutant when compared to wild-type . Data analysis revealed a significant enrichment for terms related to the phenylpropanoid metabolic process, flavonoid biosynthetic process as well as cellular amino acid derivative metabolic process. Conclusion: Our genome-wide transcriptomic analysis suggests that the ovule identity factor STK is involved in the regulation of several metabolic processes providing a strong connection between cell fate determination, development and metabolism. In particular we characterize, through phenotypic, genetic, biochemical and transcriptomic approaches, the role of STK in PAs biosynthesis. Our results indicate that STK exerts this role through the direct regulation of the gene encoding for BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This supports the theory that MADS-domain proteins control the expression of their target genes through the modification of the chromatin states. STK might recruit or negatively regulate histone modifying factors to control their activity. Moreover, we show that STK controls through a complex regulatory network not only directly BAN but also other regulators of this key gene in tannin production mRNA profiles from both Arabidopsis wild-type and stk mutant inflorescences and siliques until 5 DAP were generated by deep sequencing, in duplicate according to the manufacturer’s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5’-TGCGATGCAGAAGTTGCGCTC-3’) and RT 781 (5’-AGTACGCGGCATTGATTTCTTG-3’).
Project description:The MADS-domain transcription factor SEEDSTICK (STK) controls several aspects of plant reproduction. STK is co-expressed with CESTA (CES), a basic Helix-Loop-Helix (bHLH) transcription factor-encoding gene. CES was reported to control redundantly with the brassinosteroid positive signaling factors BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1) and BEE3 the development of the transmitting tract. Combining the stk ces-4 mutants led to a reduction in ovule fertilization due to a defect in carpel fusion which, caused the formation of holes at the center of the septum where the transmitting tract differentiates. Combining the stk mutant with the bee1 bee3 ces-4 triple mutant showed an increased number of unfertilized ovules and septum defects. The transcriptome profile of this quadruple mutant revealed a small subset of differentially expressed genes which are mainly involved in cell death, extracellular matrix and cell wall development. Our data evidence a regulatory gene network controlling transmitting tract development regulated directly or indirectly by a STK-CES containing complex and reveal new insights in the regulation of transmitting tract development by bHLH and MADS-domain transcription factors.
Project description:Like eukaryotes, bacteria express one or more serine/threonine kinases (STKs) that initiate diverse signaling networks. The STK from Streptococcus suis is encoded by a single-copy stk gene, which is crucial in stress response and virulence. To further understand the regulatory mechanism of STK in S. suis, a stk deletion strain (?stk) and its complementary strain (C?stk) were constructed to systematically decode STK characteristics by applying whole transcriptome RNA sequencing (RNA-Seq) and phosphoproteomic analysis. Numerous genes were differentially expressed in ?stk compared with the wild-type parental strain SC-19, including 320 up-regulated and 219 down-regulated genes. Particularly, 32 virulence-associated genes (VAGs) were significantly down-regulated in ?stk. Seven metabolic pathways relevant to bacterial central metabolism and translation are significantly repressed in ?stk. Phosphoproteomic analysis further identified 12 phosphoproteins that exhibit differential phosphorylation in ?stk. These proteins are associated with cell growth and division, glycolysis, and translation. Consistently, phenotypic assays confirmed that the ?stk strain displayed deficient growth and attenuated pathogenicity. Thus, STK is a central regulator that plays an important role in cell growth and division, as well as S. suis metabolism.
Project description:BACKGROUND: Chestnut rose (Rosa roxburghii Tratt) is a promising wild fruit crop in Southwest China. However, chestnut rose suffers from several important diseases such as powdery mildew and black spot. Cloning and phylogenetic analysis of plant immunity related genes will strengthen the evolutionary knowledge of plant immune system and will facilitate the utilization of candidate genes in disease resistance breeding programs. FINDINGS: Serine/threonine kinase (STK) genes, encoding one of the important proteins for defense signal transduction, were cloned from 'chestnut rose'. Fifteen STK sequences were obtained by degenerate PCR. Sequence analysis showed that nine of them have continued open reading frames, and they are separated into five classes based on sequence analysis. Interestingly, one of the classes (STK V) showed less than 40% similarity to any other class, possibly representing new type genes from chestnut rose. Southern blotting analysis revealed that the new type STK V genes are single copy, while all the other genes have several copies in the genome. Phylogenetic analysis of STK genes from chestnut rose and 21 plant species revealed that most chestnut rose genes show close relationship with Rosaceae homologs, while the STK V genes are rather ancient and form a unique clade distantly from plant homologs. CONCLUSIONS: We cloned nine STK genes from a wild fruit crop 'chestnut rose', of which a new type of STK genes was identified. The new type STK genes exist as single copies in the genome, and they are phylogenetically distant to plant homologs. The polymorphic STK genes, combined with other plant immunity genes, provide plenty of resources to be utilized to defend against pathogens attack.
Project description:The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene Fv-2, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55(P), the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk.
Project description:The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (?sse(MGAS5005)) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K(M) and k(cat) of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ?sse(MGAS5005) infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.