Functional and transcriptional analysis of chromosomal encoded hipBA type II toxin-antitoxin (TA) module from Xenorhabdus nematophila

2021 ◽  
pp. 105309
Author(s):  
Mohit Yadav ◽  
Jitendra Singh Rathore
Keyword(s):  
Transcriptional Analysis ◽  
Type Ii ◽  
Xenorhabdus Nematophila

scholarly journals Functional and Transcriptional Analysis of Chromosomal Encoded hipBAXn2 Type II Toxin-Antitoxin (TA) Module From Xenorhabdus Nematophila

2021 ◽  
Author(s):  
Mohit Yadav ◽  
Jitendra Rathore
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Transcriptional Analysis ◽  
Toxin Gene ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Xenorhabdus Nematophila ◽  
Gene Encoding ◽  
Larval Host ◽  
Adverse Environmental Conditions

Abstract Xenorhabdus nematophila is an entomopathogenic bacterium that synthesizes numerous toxins and kills its larval host. The genome of this bacterium also encodes a total of 39 putative toxin-antitoxin (TA) systems. These systems are also associated with maintaining the bacterial genomic stability and survival of bacteria under adverse environmental conditions. Three hipBA TA homologs were identified on the chromosome of X. nematophila, among them first hipBAXn TA has been studied, second hipBAXn2 TA is still unexplored while third hipBAXn3 TA has been reported as a pseudo-type TA system. Thus, for the first time, here, we are exploring the functionality of the type II hipBAXn2 TA system. This TA system was identified in the genome of X. nematophila ATCC 19061 (NCBI Refseq NC_014228) at position 3774379–3775635 bp, which consists of hipAXn2 toxin gene encoding 270 amino acid residues protein and hipBXn2 encoding antitoxin of 135 amino acid residues protein. It was observed that the overexpression of HipAXn2 toxin inhibits the growth of Escherichia coli cells in a bacteriostatic manner and amino-acids G8, H164, N167, and S169 were key residues for its toxicity. Promoter activity and expression profiling of messenger RNA from the hipBAXn2 TA system was also studied and showed that it was activated in both E. coli as well as X. nematophila upon exposure to different stress conditions. Further, we have exhibited the binding features of HipAXn2 toxin and HipBXn2 antitoxin to their promoter. This study provides the first evidence for the presence of a functional and active hipBAXn2 TA system in X. nematophila.

scholarly journals Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition duringLegionella pneumophilaIntracellular Infection of Amoebas

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00528-18 ◽  
Author(s):  
Richard C. White ◽  
Felizza F. Gunderson ◽  
Jessica Y. Tyson ◽  
Katherine H. Richardson ◽  
Theo J. Portlock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Transcriptional Analysis ◽  
Type Ii Secretion ◽  
Major Advance ◽  
Type Ii ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
First Time

ABSTRACTLegionella pneumophilagenes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection ofAcanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking eitherlapAandlapBorplaC. AplaCmutant showed even higher levels oflapAandlapBtranscripts, and alapA lapBmutant showed heightening ofplaCmRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found thatlapA plaCmutants have an ~50-fold defect during infection ofA. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of thelapA plaCmutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection ofA. castellanii. AlapA plaC nttDmutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside theLegionellagenus. LapA was notable for having as its closest homologue anA. castellaniiprotein.IMPORTANCETransmission ofL. pneumophilato humans is facilitated by its ability to grow inAcanthamoebaspecies. We previously documented that type II secretion (T2S) promotesL. pneumophilainfection ofA. castellanii. Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection ofA. castellanii. Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within theLegionellagenus in unique ways. The results of this study represent a major advance in understandingLegionellaecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.

scholarly journals Genome-scale transcriptional analysis reveals key genes associated with the development of type II diabetes in mice

2017 ◽  
Vol 13 (3) ◽  
pp. 1044-1150 ◽  
Author(s):  
Yuchi Zhang ◽  
Dongwei Han ◽  
Pengyang Yu ◽  
Qijing Huang ◽  
Pengling Ge
Keyword(s):  
Type Ii Diabetes ◽  
Transcriptional Analysis ◽  
Type Ii ◽  
Diabetes In Mice ◽  
Key Genes ◽  
Genome Scale

scholarly journals Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jitendra Singh Rathore ◽  
Lalit Kumar Gautam
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Recombinant Proteins ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Xenorhabdus Nematophila ◽  
Expression And Purification ◽  
Toxicity Assay ◽  
Neutralization Activity ◽  
E Coli

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

scholarly journals Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

2014 ◽  
Vol 197 (4) ◽  
pp. 749-761 ◽  
Author(s):  
M. A. Serbanescu ◽  
M. Cordova ◽  
K. Krastel ◽  
R. Flick ◽  
N. Beloglazova ◽  
...  
Keyword(s):  
Heat Shock ◽  
Streptococcus Mutans ◽  
Antibiotic Resistance Genes ◽  
Transcriptional Analysis ◽  
Cell Physiology ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Bacterial Physiology

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

scholarly journals Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan

2014 ◽  
Vol 80 (15) ◽  
pp. 4577-4584 ◽  
Author(s):  
Kiyotaka Fujita ◽  
Takenori Sakaguchi ◽  
Ayami Sakamoto ◽  
Michiko Shimokawa ◽  
Kanefumi Kitahara
Keyword(s):  
Bifidobacterium Longum ◽  
Transcriptional Analysis ◽  
Glycoside Hydrolase Family ◽  
Type Ii ◽  
Carbohydrate Source ◽  
Content Type ◽  
Homologous Genes ◽  
Degradative Enzymes ◽  
Bacteria And Fungi ◽  
Hydrolase Family

ABSTRACTType II arabinogalactan (AG-II) is a suitable carbohydrate source forBifidobacterium longumsubsp.longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-β-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 fromB. longumsubsp.longumJCM1217. The recombinant BLLJ_1840 expressed inEscherichia colihydrolyzed β-1,3-linked galactooligosaccharides but not β-1,4- and β-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a β-1,3-linked galactan backbone with β-1,6-linked galactan side chains. Thekcat/Kmratio of dearabinosylated LWAG was 24-fold higher than that of β-1,3-galactan. BLLJ_1840 is a novel type of exo-β-1,3-galactanase with a higher affinity for the β-1,6-substituted β-1,3-galactan than for nonsubstituted β-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo-β-1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains ofB. longumsubsp.longumandB. longumsubsp.infantisbut not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-β-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-β-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II inB. longumsubsp.longum.

Genome-wide identification, characterization, and transcriptional analysis of the metacaspase gene family in cucumber (Cucumis sativus)

Genome ◽  
2018 ◽  
Vol 61 (3) ◽  
pp. 187-194 ◽  
Author(s):  
Yong Zhou ◽  
Lifang Hu ◽  
Lunwei Jiang ◽  
Shiqiang Liu
Keyword(s):  
Gene Family ◽  
Cucumis Sativus ◽  
Stress Responses ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Plant Stress ◽  
Transcriptional Analysis ◽  
Type I ◽  
Type Ii ◽  
Motif Analysis

Metacaspase (MC), a family of caspase-like proteins, plays vital roles in regulating programmed cell death (PCD) during development and in response to stresses in plants. In this study, five MC genes (designated as CsMC1 to CsMC5) were identified in the cucumber (Cucumis sativus) genome. Sequence analysis revealed that CsMC1–CsMC3 belong to type I MC proteins, while CsMC4 and CsMC5 are type II MC proteins. Phylogenetic tree and conserved motif analysis of MC proteins indicated that these proteins can be classified into two groups, which are correlated with the types of these MC proteins. Gene structure analysis demonstrated that type I CsMC genes contain 4–7 introns, while all type II CsMC genes harbor one intron. In addition, many hormone-, stress-, and development-related cis-elements were identified in the promoter regions of CsMC genes. Expression analysis using RNA-seq data revealed that CsMC genes have distinct expression patterns in various tissues and developmental stages. qRT-PCR results showed that the transcript levels of CsMC genes could be regulated by various abiotic stresses such as NaCl, PEG, and cold. These results demonstrate that the cucumber MC gene family may function in tissue development and plant stress responses.

scholarly journals Transcriptional analysis of the endothelial response to diabetes reveals a role for galectin-3

2011 ◽  
Vol 43 (20) ◽  
pp. 1144-1152 ◽  
Author(s):  
April L. Darrow ◽  
Ralph V. Shohet ◽  
J. Gregory Maresh
Keyword(s):  
Type Ii Diabetes ◽  
Transcriptional Response ◽  
Protein Quantification ◽  
Transcriptional Analysis ◽  
Type Ii ◽  
Transcriptional Responses ◽  
Endothelial Response ◽  
Galectin 3 ◽  
Normal Chow ◽  
Inflammatory State

To characterize the endothelial dysfunction associated with Type II diabetes, we surveyed transcriptional responses in the vascular endothelia of mice receiving a diabetogenic, high-fat diet. Tie2-GFP mice were fed a diet containing 60% fat calories (HFD); controls were littermates fed normal chow. Following 4, 6, and 8 wk, aortic and leg muscle tissues were enzymatically dispersed, and endothelial cells were obtained by fluorescence-activated cell sorting. Relative mRNA abundance in HFD vs. control endothelia was measured with long-oligo microarrays; highly dysregulated genes were confirmed by real-time PCR and protein quantification. HFD mice were hyperglycemic by 2 wk and displayed vascular insulin resistance and decreased glucose tolerance by 5 and 6 wk, respectively. Endothelial transcripts upregulated by HFD included galectin-3 ( Lgals3), 5-lipoxygenase-activating protein, and chemokine ligands 8 and 9. Increased LGALS3 protein was detected in muscle endothelium by immunohistology accompanied by elevated LGALS3 in the serum of HFD mice. Our comprehensive analysis of the endothelial transcriptional response in a model of Type II diabetes reveals novel regulation of transcripts with roles in inflammation, insulin sensitivity, oxidative stress, and atherosclerosis. Increased endothelial expression and elevated humoral levels of LGALS3 supports a role for this molecule in the vascular response to diabetes, and its potential as a direct biomarker for the inflammatory state in diabetes.

scholarly journals Transcriptional Analysis and Functional Characterization of a Gene Pair Encoding Iron-Regulated Xenocin and Immunity Proteins of Xenorhabdus nematophila

2008 ◽  
Vol 190 (11) ◽  
pp. 3877-3885 ◽  
Author(s):  
Jitendra Singh ◽  
Nirupama Banerjee
Keyword(s):  
Bacterial Species ◽  
Functional Characterization ◽  
Inducible Promoter ◽  
Transcriptional Analysis ◽  
Transcriptional Unit ◽  
Xenorhabdus Nematophila ◽  
Immunity Protein ◽  
E Coli ◽  
Larval Stages

ABSTRACT We describe a two-gene cluster encoding a bacteriocin, xenocin, and the cognate immunity protein in the insect-pathogenic bacterium Xenorhabdus nematophila, which infects and kills larval stages of the common crop pest Helicoverpa armigera. The two genes, xcinA and ximB, are present in the genome as a single transcriptional unit, which is regulated under SOS conditions. The stress-inducible promoter was activated by mitomycin C, glucose, and Fe3+ depletion and at an elevated temperature when it was tested in Escherichia coli cells. Expression of the xenocin protein alone in E. coli inhibited the growth of this organism. The growth inhibition was abolished when the immunity protein was also present. A recombinant xenocin-immunity protein complex inhibited the growth of E. coli indicator cells when it was added exogenously to a growing culture. Xenocin is an endoribonuclease with an enzymatically active C-terminal domain. Six resident bacterial species (i.e., Bacillus, Enterobacter, Enterococcus, Citrobacter, Serratia, and Stenotrophomonas species) from the H. armigera gut exhibited sensitivity to recombinant xenocin when the organisms were grown under iron-depleted conditions and at a high temperature. Xenocin also inhibited the growth of two Xenorhabdus isolates. This study demonstrates that Fe3+ depletion acts as a common cue for synthesis of xenocin by X. nematophila and sensitization of the target strains to the bacteriocin.

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