scholarly journals Regulation of Resistance to Copper in Xanthomonas axonopodis pv. vesicatoria

2005 ◽  
Vol 71 (2) ◽  
pp. 782-789 ◽  
Author(s):  
Andreas E. Voloudakis ◽  
Therese M. Reignier ◽  
Donald A. Cooksey
Keyword(s):  
Pseudomonas Syringae ◽  
Transcriptional Activation ◽  
Genomic Sequence ◽  
Direct Interaction ◽  
Protein Product ◽  
Inducible Expression ◽  
Copper Resistance ◽  
Start Codon ◽  
Xanthomonas Axonopodis ◽  
E Coli

ABSTRACT Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the cop and pco operons of Pseudomonas syringae and Escherichia coli, respectively. However, instead of the two-component (copRS and pcoRS) systems determining copper-inducible expression of the operons in P. syringae and E. coli, a novel open reading frame, copL, was found to be required for copper-inducible expression of the downstream multicopper oxidase copA in X. axonopodis. copL encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within copL, as well as an amino acid substitution generated at the putative start codon of copL, caused a loss of copper-inducible transcriptional activation of copA. A nonpolar insertion of a kanamycin resistance gene in copL resulted in copper sensitivity in the wild-type strain. However, repeated attempts to complement copL mutations in trans failed. Analysis of the genomic sequence databases shows that there are copL homologs upstream of copAB genes in X. axonopodis pv. citri, X. campestris pv. campestris, and Xylella fastidiosa. The cloned promoter area upstream of copA in X. axonopodis pv. vesicatoria did not function in Pseudomonas syringae or in E. coli, nor did the P. syringae cop promoter function in Xanthomonas. However, a transcriptional fusion of the Xanthomonas cop promoter with the Pseudomonas copABCDRS was able to confer resistance to copper in Xanthomonas, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria.

scholarly journals Genome rearrangements induce biofilm formation inEscherichia coliC – an old model organism with a new application in biofilm research

2019 ◽  
Author(s):  
Jarosław E. Król ◽  
Donald C. Hall ◽  
Sergey Balashov ◽  
Steven Pastor ◽  
Justin Siebert ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Insertion Sequence ◽  
Genomic Sequence ◽  
Expression Profiles ◽  
Model Organism ◽  
The Other ◽  
Start Codon ◽  
Bacterial Survival ◽  
E Coli ◽  
Full Genomic Sequence

AbstractEscherichia coliC forms more robust biofilms than the other laboratory strains. Biofilm formation and cell aggregation under a high shear force depends on temperature and salt concentrations. It is the last of fiveE. colistrains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43,waaSBOJYZULfor LPS synthesis, andcpsBfor curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulatorcsrAgene. This insertion is located 86 bp upstream of thecsrAstart codon inside the −35 region of P4 promoter and blocks the transcription from the sigma32and sigma70promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of thecsgDgene, which may drive its overexpression in biofilm. And finally,E. coliC encodes an additional sigma70subunit overexpressed in biofilm and driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions and total expression profiles using RNA-seq analyses comparing planktonic and biofilm envirovars provided insights into understanding this regulatory pathway inE. coli.IMPORTANCEBiofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains ofE. coligrown for decadesin vitrohave evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain ofE. coliC produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence as well as gene expression profiles of both the biofilm and planktonic envirovars of this classic strain, which provide for a base level of characterization and make it useful for many biofilm-based applications.

scholarly journals Genome rearrangements induce biofilm formation in Escherichia coli C – an old model organism with a new application in biofilm research

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jarosław E. Król ◽  
Donald C. Hall ◽  
Sergey Balashov ◽  
Steven Pastor ◽  
Justin Sibert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Insertion Sequence ◽  
Genomic Sequence ◽  
Model Organism ◽  
Start Codon ◽  
Bacterial Survival ◽  
E Coli ◽  
Full Genomic Sequence

Abstract Background Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Results Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the − 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. Conclusions Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

scholarly journals Identification of a Copper-Responsive Two-Component System on the Chromosome of Escherichia coli K-12

2000 ◽  
Vol 182 (20) ◽  
pp. 5864-5871 ◽  
Author(s):  
George P. Munson ◽  
Deborah L. Lam ◽  
F. Wayne Outten ◽  
Thomas V. O'Halloran
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Metal Ion ◽  
Copper Ions ◽  
Copper Ion ◽  
Regulatory System ◽  
Copper Resistance ◽  
E Coli ◽  
Two Component ◽  
K 12

ABSTRACT Using a genetic screen we have identified two chromosomal genes,cusRS (ylcA ybcZ), from Escherichia coli K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of pcoE, a plasmid-borne gene from the E. coli copper resistance operon pco. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the pcooperon of E. coli; CopR and CopS from thecop operon, which provides copper resistance toPseudomonas syringae; and SilR and SilS from thesil locus, which provides silver ion resistance toSalmonella enterica serovar Typhimurium. The genescusRS are also required for the copper-dependent expression of at least one chromosomal gene, designated cusC(ylcB), which is allelic to the recently identified virulence gene ibeB in E. coli K1. Thecus locus may comprise a copper ion efflux system, because the expression of cusC is induced by high concentrations of copper ions. Furthermore, the translation products of cusCand additional downstream genes are homologous to known metal ion antiporters.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals Construction and Characterization of a proU-gfp Transcriptional Fusion That Measures Water Availability in a Microbial Habitat

2002 ◽  
Vol 68 (9) ◽  
pp. 4604-4612 ◽  
Author(s):  
Catherine A. Axtell ◽  
Gwyn A. Beattie
Keyword(s):  
Pseudomonas Syringae ◽  
Water Availability ◽  
Water Deprivation ◽  
Bacterial Species ◽  
Transcriptional Fusion ◽  
Bacterial Biosensor ◽  
E Coli ◽  
Quantitative Manner ◽  
Microbial Habitat

ABSTRACT We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the proU promoter from Escherichia coli and the reporter gene gfp was introduced into strains of E. coli, Pantoea agglomerans, and Pseudomonas syringae. The proU-gfp fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na2SO4, KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced proU-gfp expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of P. agglomerans biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a proU-gfp-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of proU-gfp in multiple bacterial species illustrates the potential for tailoring proU-gfp-based biosensors to specific habitats.

scholarly journals Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria

2005 ◽  
Vol 71 (12) ◽  
pp. 8284-8291 ◽  
Author(s):  
Huseyin Basim ◽  
Gerald V. Minsavage ◽  
Robert E. Stall ◽  
Jaw-Fen Wang ◽  
Savita Shanker ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Resistance Genes ◽  
Xanthomonas Campestris ◽  
Sinorhizobium Meliloti ◽  
The United States ◽  
Copper Resistance ◽  
Pcr Analysis ◽  
Pepper Plant ◽  
Gene Encoding ◽  
Copper Resistance Genes

ABSTRACT We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.

scholarly journals Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

2010 ◽  
Vol 76 (8) ◽  
pp. 2500-2508 ◽  
Author(s):  
S. D. Braun ◽  
J. Hofmann ◽  
A. Wensing ◽  
M. S. Ullrich ◽  
H. Weingart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Pentanoic Acid ◽  
Promising Candidate ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Highly Active ◽  
Putative Aminotransferase

ABSTRACT The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km , 7 mM; k cat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

scholarly journals CTRP9 Mediates Protective Effects in Cardiomyocytes via AMPK- and Adiponectin Receptor-Mediated Induction of Anti-Oxidant Response

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1229
Author(s):  
Bernd Niemann ◽  
Ling Li ◽  
Dorothee Siegler ◽  
Benedikt H. Siegler ◽  
Fabienne Knapp ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Direct Interaction ◽  
Left Ventricular ◽  
Cardiac Cells ◽  
Adiponectin Receptor ◽  
Protective Effects ◽  
Necrosis Factor Alpha ◽  
Anti Oxidant ◽  
Cardioprotective Effects ◽  
The Right

The C1q/tumor necrosis factor-alpha-related protein 9 (CTRP9) has been reported to exert cardioprotective effects, but its role in the right ventricle (RV) remains unclear. To investigate the role of CTRP9 in RV hypertrophy and failure, we performed pulmonary artery banding in weanling rats to induce compensatory RV hypertrophy seven weeks after surgery and RV failure 22 weeks after surgery. CTRP9 expression, signal transduction and mechanisms involved in protective CTRP9 effects were analyzed in rat and human RV tissue and cardiac cells. We demonstrate that CTRP9 was induced during compensatory RV hypertrophy but almost lost at the stage of RV failure. RV but not left ventricular (LV) cardiomyocytes or RV endothelial cells demonstrated increased intracellular reactive oxygen species (ROS) and apoptosis activation at this stage. Exogenous CTRP9 induced AMP-activated protein kinase (AMPK)-dependent transcriptional activation of the anti-oxidant thioredoxin-1 (Trx1) and superoxide dismutase-2 (SOD2) and reduced phenylephrine-induced ROS. Combined knockdown of adiponectin receptor-1 (AdipoR1) and AdipoR2 or knockdown of calreticulin attenuated CTRP9-mediated anti-oxidant effects. Immunoprecipitation showed an interaction of AdipoR1 with AdipoR2 and the co-receptor T-cadherin, but no direct interaction with calreticulin. Thus, CTRP9 mediates cardioprotective effects through inhibition of ROS production induced by pro-hypertrophic agents via AMPK-mediated activation of anti-oxidant enzymes.

scholarly journals Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

BioTech ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 24
Author(s):  
Marina Snapyan ◽  
Sylvain Robin ◽  
Garabet Yeretssian ◽  
Michèle Lecocq ◽  
Frédéric Marc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genomic Sequence ◽  
Synthetic Activity ◽  
Translation System ◽  
Free System ◽  
Transcription Terminator ◽  
Cell Free System ◽  
E Coli ◽  
Bacterial Transcription ◽  
A Cell

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

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