scholarly journals Hydrophobic mismatch effect is a key factor in protein transport on the Tat pathway

2021 ◽  
Author(s):  
Binhan Hao ◽  
Wenjie Zhou ◽  
Steven M Theg
Keyword(s):  
Protein Transport ◽  
Bacterial Species ◽  
Plant Mitochondria ◽  
Membrane Integrity ◽  
Critical Threshold ◽  
Hydrophobic Mismatch ◽  
Membrane Bilayer ◽  
E Coli ◽  
Tat Pathway ◽  
Folded Proteins

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we access the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

scholarly journals Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria

2020 ◽  
Author(s):  
Bationa Bennewitz ◽  
Mayank Sharma ◽  
Franzisca Tannert ◽  
Ralf Bernd Klösgen
Keyword(s):  
Plant Mitochondria ◽  
Common Mechanism ◽  
Twin Arginine Translocation ◽  
Dual Targeting ◽  
In Organello ◽  
Highly Sensitive ◽  
Tat Pathway ◽  
Electron Transport Chains ◽  
Membrane Bound ◽  
Folded Proteins

AbstractThe biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA. Here we show that the nuclearly encoded TatA has dual targeting properties, i.e., it can be imported into both, chloroplasts and mitochondria. Dual targeting of TatA was observed with in organello experiments employing isolated chloroplasts and mitochondria as well as after transient expression of suitable reporter constructs in epidermal leaf cells. The extent of transport of these constructs into mitochondria of transiently transformed leaf cells was relatively low, causing a demand for highly sensitive methods to be detected, like the sasplitGFP approach. Yet, the dual import of TatA into mitochondria and chloroplasts observed here points to a common mechanism of Tat transport for folded proteins within both endosymbiotic organelles.

scholarly journals Aminoglycosides induce a bacterial senescent state that increases antibiotic tolerance in treatment-naïve cells

2021 ◽  
Author(s):  
Christian T. Meyer ◽  
Giancarlo N. Bruni ◽  
Ben Dodd ◽  
Joel M. Kralj
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacterial Species ◽  
Genetic Resistance ◽  
Membrane Integrity ◽  
Antibiotic Tolerance ◽  
Paracrine Signaling ◽  
E Coli ◽  
Evolutionary Innovation ◽  
Treatment Naïve

Bacterial evolution of antibiotic resistance is facilitated by non-genetic resistance that increases drug tolerance, buying time for evolutionary innovation. Escherichia coli treated with aminoglycosides permanently lose the ability to divide within four hours, yet we discovered a majority of cells maintain membrane integrity and metabolic activity greater than two days post treatment — a bacterial senescent-like state. These cells, which we term zombies, exhibit dynamic gene expression and metabolomic profiles, even after irreversible exit from the cell cycle. Our data reveal zombies upregulate the phage shock protein pathway to maintain membrane integrity. Remarkably, though unable to form new colonies, zombies increase the antibiotic tolerance of treatment-naïve cells, implying chemical communication. Chemical supplementation and genetic knockouts show that zombies communicate with treatment-naïve cells by secreting indole. In summary, our study revealed a bacterial senescent-like state, induced by aminoglycosides, that decreases the antibiotic susceptibility of multiple bacterial species. Thus, E. coli zombies utilize paracrine signaling to promote non-genetic antibiotic tolerance.

scholarly journals Twin-Arginine Translocation Pathway inStreptomyces lividans

2001 ◽  
Vol 183 (23) ◽  
pp. 6727-6732 ◽  
Author(s):  
Kristien Schaerlaekens ◽  
Michaela Schierová ◽  
Elke Lammertyn ◽  
Nick Geukens ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Chimeric Construct ◽  
Translocation Pathway ◽  
Folded Proteins ◽  
Subtilisin Inhibitor ◽  
Dependent Transport

ABSTRACT The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in theS. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, atatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticustyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividansbut not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general inStreptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.

scholarly journals Specificity of Signal Peptide Recognition in Tat-Dependent Bacterial Protein Translocation

2001 ◽  
Vol 183 (2) ◽  
pp. 604-610 ◽  
Author(s):  
Natascha Blaudeck ◽  
Georg A. Sprenger ◽  
Roland Freudl ◽  
Thomas Wiegert
Keyword(s):  
Fusion Protein ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Signal Peptides ◽  
E Coli ◽  
Amino Terminal ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Dependent Protein ◽  
Folded Proteins

ABSTRACT The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) ofZymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC andtatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

Efficacy of Silver Nanoparticles Synthesized from Hymenocallis species (Spider Lilly) Leaf Extract as Antimicrobial Agents

2019 ◽  
Vol 12 (5) ◽  
pp. 4644-4647
Author(s):  
K.K. Gupta ◽  
Neha Kumari ◽  
Neha Sinha ◽  
Akruti Gupta
Keyword(s):  
Silver Nanoparticles ◽  
Leaf Extract ◽  
Antimicrobial Agents ◽  
Color Change ◽  
Bacterial Species ◽  
Antimicrobial Property ◽  
Biogenic Synthesis ◽  
E Coli ◽  
Light Yellow ◽  
Antibiotic Property

Biogenic synthesis of silver nanoparticles synthesized from Hymenocallis species (Spider Lilly) leaf extract was subjected for investigation of its antimicrobial property against four bacterial species (E. coli, Salmonella sp., Streptococcus sp. & Staphylococcus sp.). The results revealed that synthesized nanoparticles solution very much justify the color change property from initial light yellow to final reddish brown during the synthesis producing a characteristics absorption peak in the range of 434-466 nm. As antimicrobial agents, their efficacy was evaluated by analysis of variance in between the species and among the different concentration of AgNPs solution, which clearly showed that there was significant variation in the antibiotic property between the four different concentrations of AgNPs solution and also among four different species of bacteria taken under studies. However, silver nanoparticles solution of 1: 9 and 1:4 were proved comparatively more efficient as antimicrobial agents against four species of bacteria.

Substituted 6,7-dimethoxy-5-oxo-2,3,5,9b-tetrahydrothiazolo[2,3-a]isoindole- 3-1,1-dioxide Derivatives with Antimicrobial Activity and Docking Assisted Prediction of the Mechanism of their Antibacterial and Antifungal Properties

2020 ◽  
Vol 20 (29) ◽  
pp. 2681-2691
Author(s):  
Athina Geronikaki ◽  
Victor Kartsev ◽  
Phaedra Eleftheriou ◽  
Anthi Petrou ◽  
Jasmina Glamočlija ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antifungal Activity ◽  
Drug Targets ◽  
Bacterial Species ◽  
Pharmaceutical Research ◽  
Docking Studies ◽  
Fungal Species ◽  
Docking Analysis ◽  
E Coli ◽  
Antibacterial And Antifungal

Background: Although a great number of the targets of antimicrobial therapy have been achieved, it remains among the first fields of pharmaceutical research, mainly because of the development of resistant strains. Docking analysis may be an important tool in the research for the development of more effective agents against specific drug targets or multi-target agents 1-3. Methods: In the present study, based on docking analysis, ten tetrahydrothiazolo[2,3-a]isoindole derivatives were chosen for the evaluation of the antimicrobial activity. Results: All compounds showed antibacterial activity against eight Gram-positive and Gram-negative bacterial species being, in some cases, more potent than ampicillin and streptomycin against all species. The most sensitive bacteria appeared to be S. aureus and En. Cloacae, while M. flavus, E. coli and P. aeruginosa were the most resistant ones. The compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited good antifungal activity better than reference drugs bifonazole (1.4 – 41 folds) and ketoconazole (1.1 – 406 folds) against all fungal species. In order to elucidate the mechanism of action, docking studies on different antimicrobial targets were performed. Conclusion: According to docking analysis, the antifungal activity can be explained by the inhibition of the CYP51 enzyme for most compounds with a better correlation of the results obtained for the P.v.c. strain (linear regression between estimated binding Energy and log(1/MIC) with R 2 =0.867 and p=0.000091 or R 2 = 0.924, p= 0.000036, when compound 3 is excluded.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Characterization of Escherichia coli Strains Derived from Cow Milk of Subclinical and Clinical Cases of Mastitis

2021 ◽  
Vol 11 (2) ◽  
pp. 541
Author(s):  
Katarzyna Grudlewska-Buda ◽  
Krzysztof Skowron ◽  
Ewa Wałecka-Zacharska ◽  
Natalia Wiktorczyk-Kapischke ◽  
Jarosław Bystroń ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
Subclinical Mastitis ◽  
Clinical Mastitis ◽  
Economic Problem ◽  
Cow Milk ◽  
P Value ◽  
Dairy Herds ◽  
E Coli ◽  
Bonferroni Test

Mastitis is a major economic problem in dairy herds, as it might decrease fertility, and negatively affect milk quality and milk yield. Out of over 150 bacterial species responsible for the udder inflammation, Escherichia coli is one of the most notable. This study aimed to assess antimicrobial susceptibility, resistance to dipping agents and biofilm formation of 150 E. coli strains isolated from milk of cows with subclinical and clinical mastitis. The strains came from three dairy herds located in Northern and Central Poland. The statistical analyses were performed with post-hoc Bonferroni test and chi-square test (including Yates correction). The data with a p value of <0.05 were considered significant. We found that the tested strains were mostly sensitive to antimicrobials and dipping agents. It was shown that 37.33% and 4.67% of strains were resistant and moderately resistant to at least one antimicrobial agent, respectively. No extended-spectrum beta-lactamases (ESBL)-producing E. coli were detected. The majority of strains did not possess the ability to form biofilm or formed a weak biofilm. The strong biofilm formers were found only among strains derived from cows with subclinical mastitis. The lowest bacteria number was noted for subclinical mastitis cows’ strains, after stabilization with iodine (3.77 log CFU × cm−2) and chlorhexidine (3.96 log CFU × cm−2) treatment. In the present study, no statistically significant differences in susceptibility to antibiotics and the ability to form biofilm were found among the strains isolated from cows with subclinical and clinical mastitis. Despite this, infections in dairy herds should be monitored. Limiting the spread of bacteria and characterizing the most common etiological factors would allow proper treatment.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

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