Expression and Function of clpS and clpA in Xanthomonas Campestris Pv. Campestris

Abstract ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in many regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen and can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris have several clp genes, namely clpS, clpA, clpX, clpP, clpQ, and clpY. Among them, only clpX and clpP were known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex, respectively. Transcriptional analysis revealed that expression of both clpS and clpA is induced by heat shock. Inactivation of clpA but not clpS resulted in susceptibility to high temperature and revealed an attenuation of virulence on the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 are critical amino acid residues for ClpA function in heat tolerance. The clpA mutant revealed different protein expression profiles as compared to the wild type in response to heat stress. Taken together, we characterized two clp genes (clpS and clpA) by examining their expression and function including stress tolerance and pathogenicity. We demonstrated that both clpS and clpA are expressed in a temperature dependent manner, and clpA is required for survival at high temperature and full virulence of X. campestris pv. campestris. This is the first time that clpS and clpA has been characterized in Xanthomonas.

Download Full-text

Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

Microbiology ◽

10.1099/mic.0.27598-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 597-605 ◽

Cited By ~ 23

Author(s):

Nopmanee Chauvatcharin ◽

Sopapan Atichartpongkul ◽

Supa Utamapongchai ◽

Wirongrong Whangsuk ◽

Paiboon Vattanaviboon ◽

...

Keyword(s):

Xanthomonas Campestris ◽

Stationary Phases ◽

Transcriptional Analysis ◽

Dependent Manner ◽

Mobility Shift ◽

Highly Sensitive ◽

Organic Hydroperoxides ◽

Physiological Analysis ◽

Gel Mobility Shift ◽

A Minor

katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA–ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like σ 70 −35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.

Download Full-text

Identification of Proteins Differentially Expressed by Adipose-derived Mesenchymal Stem Cells Isolated from Immunodeficient Mice

International Journal of Molecular Sciences ◽

10.3390/ijms20112672 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2672 ◽

Cited By ~ 1

Author(s):

Yoshiki Nakashima ◽

Saifun Nahar ◽

Chika Miyagi-Shiohira ◽

Takao Kinjo ◽

Naoya Kobayashi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Expression Profiles ◽

Severe Combined Immunodeficiency ◽

Therapeutic Effects ◽

Immunodeficient Mice ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

Protein Expression Profiles ◽

And Function

Although cell therapy using adipose-derived mesenchymal stem cells (AdMSCs) regulates immunity, the degree to which cell quality and function are affected by differences in immunodeficiency of donors is unknown. We used liquid chromatography tandem-mass spectrometry (LC MS/MS) to identify the proteins expressed by mouse AdMSCs (mAsMSCs) isolated from normal (C57BL/6) mice and mice with severe combined immunodeficiency (SCID). The protein expression profiles of each strain were 98%–100% identical, indicating that the expression levels of major proteins potentially associated with the therapeutic effects of mAdMSCs were highly similar. Further, comparable levels of cell surface markers (CD44, CD90.2) were detected using flow cytometry or LC MS/MS. MYH9, ACTN1, CANX, GPI, TPM1, EPRS, ITGB1, ANXA3, CNN2, MAPK1, PSME2, CTPS1, OTUB1, PSMB6, HMGB1, RPS19, SEC61A1, CTNNB1, GLO1, RPL22, PSMA2, SYNCRIP, PRDX3, SAMHD1, TCAF2, MAPK3, RPS24, and MYO1E, which are associated with immunity, were expressed at higher levels by the SCID mAdMSCs compared with the C57BL/6 mAdMSCs. In contrast, ANXA9, PCBP2, LGALS3, PPP1R14B, and PSMA6, which are also associated with immunity, were more highly expressed by C57BL/6 mAdMSCs than SCID mAdMSCs. These findings implicate these two sets of proteins in the pathogenesis and maintenance of immunodeficiency.

Download Full-text

Anterior Hippocampus in Schizophrenia Pathogenesis: Molecular Evidence from a Proteome Study

Australian & New Zealand Journal of Psychiatry ◽

10.1080/00048670902721103 ◽

2009 ◽

Vol 43 (4) ◽

pp. 310-322 ◽

Cited By ~ 29

Author(s):

Maryam Nesvaderani ◽

Izuru Matsumoto ◽

Sinthuja Sivagnanasundaram

Keyword(s):

Mitochondrial Function ◽

Gel Electrophoresis ◽

Expression Profiles ◽

Structure And Function ◽

Differentially Expressed ◽

Molecular Evidence ◽

Differentially Expressed Proteins ◽

Fresh Frozen ◽

Protein Expression Profiles ◽

And Function

Objective: The purpose of the present study was to identify differentially expressed proteins in the anterior and posterior hippocampus of brains of schizophrenia patients compared to neurologically healthy controls. Method: Proteins extracted from fresh frozen post-mortem posterior and anterior hippocampus for nine schizophrenia and nine control individuals, and seven schizophrenia and seven control individuals, respectively, were screened for differential expression using 2-D gel electrophoresis and mass spectrometry. Results: A significantly larger number of protein spots were differentially expressed in the anterior (n = 43) compared to the posterior (n = 16) hippocampus, representing 34 and 14 unique proteins, respectively. These proteins are involved in cytoskeleton structure and function, neurotransmission and mitochondrial function. Conclusion: Based on the aberrant protein expression profiles, the anterior hippocampus appears to be more involved in schizophrenia pathogenesis than the posterior hippocampus. Furthermore, consistent with previous findings, we found molecular evidence to support abnormal neuronal cytoarchitecture and function, neurotransmission and mitochondrial function in the schizophrenia hippocampus.

Download Full-text

New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

Scientific Reports ◽

10.1038/s41598-021-83240-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sara Bitam ◽

Ahmad Elbahnsi ◽

Geordie Creste ◽

Iwona Pranke ◽

Benoit Chevalier ◽

...

Keyword(s):

Phosphinic Acid ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Transmembrane Conductance Regulator ◽

Intracellular Loop ◽

Respiratory Cells ◽

Cftr Protein ◽

Dynamics Simulations ◽

And Function ◽

Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

Download Full-text

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

Biochemistry and Cell Biology ◽

10.1139/o97-079 ◽

1997 ◽

Vol 75 (6) ◽

pp. 687-696 ◽

Cited By ~ 20

Author(s):

Tamo Fukamizo ◽

Ryszard Brzezinski

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Structure And Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Streptomyces Sp ◽

Binding Cleft ◽

And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

Download Full-text

A Combined Metabolomic and Proteomic Study Revealed the Difference in Metabolite and Protein Expression Profiles in Ruminal Tissue From Goats Fed Hay or High-Grain Diets

Frontiers in Physiology ◽

10.3389/fphys.2019.00066 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 4

Author(s):

Changzheng Guo ◽

Daming Sun ◽

Xinfeng Wang ◽

Shengyong Mao

Keyword(s):

Protein Expression ◽

Expression Profiles ◽

Proteomic Study ◽

The Difference ◽

Protein Expression Profiles

Download Full-text

Gene Expression and Transcriptome Profiling of Changes in a Cancer Cell Line Post-Exposure to Cadmium Telluride Quantum Dots: Possible Implications in Oncogenesis

Dose-Response ◽

10.1177/15593258211019880 ◽

2021 ◽

Vol 19 (2) ◽

pp. 155932582110198

Author(s):

Mohammed S. Aldughaim ◽

Mashael R. Al-Anazi ◽

Marie Fe F. Bohol ◽

Dilek Colak ◽

Hani Alothaid ◽

...

Keyword(s):

Gene Expression ◽

Quantum Dots ◽

Cancer Cells ◽

Cadmium Telluride ◽

Cancer Progression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Dependent Manner ◽

Cdte Qds ◽

Cadmium Telluride Quantum Dots

Cadmium telluride quantum dots (CdTe-QDs) are acquiring great interest in terms of their applications in biomedical sciences. Despite earlier sporadic studies on possible oncogenic roles and anticancer properties of CdTe-QDs, there is limited information regarding the oncogenic potential of CdTe-QDs in cancer progression. Here, we investigated the oncogenic effects of CdTe-QDs on the gene expression profiles of Chang cancer cells. Chang cancer cells were treated with 2 different doses of CdTe-QDs (10 and 25 μg/ml) at different time intervals (6, 12, and 24 h). Functional annotations helped identify the gene expression profile in terms of its biological process, canonical pathways, and gene interaction networks activated. It was found that the gene expression profiles varied in a time and dose-dependent manner. Validation of transcriptional changes of several genes through quantitative PCR showed that several genes upregulated by CdTe-QD exposure were somewhat linked with oncogenesis. CdTe-QD-triggered functional pathways that appear to associate with gene expression, cell proliferation, migration, adhesion, cell-cycle progression, signal transduction, and metabolism. Overall, CdTe-QD exposure led to changes in the gene expression profiles of the Chang cancer cells, highlighting that this nanoparticle can further drive oncogenesis and cancer progression, a finding that indicates the merit of immediate in vivo investigation.

Download Full-text

Comparison of gene expression in the red imported fire ant (Solenopsis invicta) under different temperature conditions

Scientific Reports ◽

10.1038/s41598-021-95779-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mohammad Vatanparast ◽

Robert T. Puckett ◽

Deuk-Soo Choi ◽

Youngjin Park

Keyword(s):

Gene Expression ◽

Gene Ontology ◽

High Temperature ◽

Temperature Stress ◽

Solenopsis Invicta ◽

Expression Profiles ◽

Fire Ant ◽

Molecular Response ◽

Red Imported Fire Ant ◽

Imported Fire Ant

AbstractThe red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and is known as a global problematic invasive species. This study focused on the molecular response of RIFA by comparing gene expression profiles after exposing ants to low (10 °C) and high (40 °C) temperature stress and comparing them to untreated controls (30 °C). A total of 99,085 unigenes (the clustered non-redundant transcripts that are filtered from the longest assembled contigs) were obtained, of which 19,154 were annotated with gene descriptions, gene ontology terms, and metabolic pathways. 86 gene ontology (GO) functional sub-groups and 23 EggNOG terms resulted. Differentially expressed genes (DEGs) with log2FC ≥ 10 were screened and were compared at different temperatures. We found 203, 48, and 66 specific DEGs co-regulated at 10, 20, and 40 °C. Comparing transcriptome profiles for differential gene expression resulted in various DE genes, including cytochrome P450, NADH dehydrogenase subunit 1, cuticle protein and heat shock protein (HSP), which have previously been reported to be involved in cold and high temperature resistance. GO analysis revealed that antioxidant activity is up-regulated under high temperature stress. We verified the RNA-seq data by qPCR on 20 up- and down-regulated DEGs. These findings provide a basis for future understanding of the adaptation mechanisms of RIFA and the molecular mechanisms underlying the response to low and high temperatures.

Download Full-text