scholarly journals Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (10) ◽  
pp. 1635-1647 ◽  
Author(s):  
Lars-Göran Ottosson ◽  
Katarina Logg ◽  
Sebastian Ibstedt ◽  
Per Sunnerhagen ◽  
Mikael Käll ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Knockout ◽  
Common Mechanism ◽  
Sulfate Assimilation ◽  
Complete Collection ◽  
Content Type ◽  
Knockout Mutants ◽  
Sulfate Assimilation Pathway ◽  
Mechanistic Basis ◽  
Respiratory Mechanism

ABSTRACT Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.

scholarly journals The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

2012 ◽  
Vol 56 (6) ◽  
pp. 3216-3223 ◽  
Author(s):  
Tina Manzhu Kang ◽  
Jessica Yuan ◽  
Angelyn Nguyen ◽  
Elinne Becket ◽  
Hanjing Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Gene Knockout ◽  
Aminoglycoside Antibiotic ◽  
Double Strand Breaks ◽  
Repair Capacity ◽  
Content Type ◽  
Strand Breaks ◽  
Knockout Mutants ◽  
Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

scholarly journals FLO11Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

2013 ◽  
Vol 79 (19) ◽  
pp. 6023-6032 ◽  
Author(s):  
Marc Bou Zeidan ◽  
Lourdes Carmona ◽  
Severino Zara ◽  
Jose F. Marcos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Gene Knockout ◽  
Yeast Cells ◽  
Knockout Mutant ◽  
Fungal Cell ◽  
Content Type ◽  
Cell Penetrating ◽  
Flor Yeasts

ABSTRACTSaccharomyces cerevisiae“flor” yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends onFLO11gene length and expression.FLO11encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm ofS. cerevisiaeflor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functionalFLO11but was not accompanied by increasedFLO11expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that aflo11gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that theFLO11gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

scholarly journals Draft Genome Sequences of Salmonella Lysozyme Gene Knockout Mutants

2017 ◽  
Vol 5 (23) ◽  
Author(s):  
Narine Arabyan ◽  
Bihua C. Huang ◽  
Bart C. Weimer
Keyword(s):  
Gene Deletion ◽  
Gene Knockout ◽  
Draft Genome ◽  
Deletion Mutants ◽  
Genome Sequences ◽  
Content Type ◽  
Knockout Mutants ◽  
Serovar Typhimurium ◽  
Encoding Gene ◽  
Glucoside Hydrolases

ABSTRACT Lysozyme enzymes hydrolyze the β-1,4-glycosidic bond in oligosaccharides. These enzymes are part of a broad group of glucoside hydrolases that are poorly characterized; however, they are important for growth and are being recognized as emerging virulence factors. This is the release of four lysozyme-encoding-gene-deletion mutants in Salmonella enterica serovar Typhimurium LT2.

scholarly journals Environmental Calcium Initiates a Feed-Forward Signaling Circuit That Regulates Biofilm Formation and Rugosity inVibrio vulnificus

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Daniel M. Chodur ◽  
Patrick Coulter ◽  
Jacob Isaacs ◽  
Meng Pu ◽  
Nico Fernandez ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Second Messenger ◽  
Sulfate Assimilation ◽  
Human Pathogen ◽  
Metabolic Intermediate ◽  
Feed Forward ◽  
Content Type ◽  
Environmental Signals ◽  
Environmental Signal ◽  
Sulfate Assimilation Pathway

ABSTRACTPoor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogenVibrio vulnificus.V. vulnificus, as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of thebrpextracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-inducedPbrpAexpression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), was required for calcium-inducedPbrpAexpression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger inV. vulnificus. Moreover, c-di-GMP and BrpT (the activator ofbrpexpression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boostbrpexpression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation ofV. vulnificusbiofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism.IMPORTANCEThe second messenger c-di-GMP is a key regulator of bacterial physiology. TheV. vulnificusgenome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation inV. vulnificus. Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggersbrp-mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.

Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae

2012 ◽  
Vol 94 (5) ◽  
pp. 1313-1319 ◽  
Author(s):  
Kiyotaka Y. Hara ◽  
Kentaro Kiriyama ◽  
Akiko Inagaki ◽  
Hideki Nakayama ◽  
Akihiko Kondo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Sulfate Assimilation ◽  
Sulfate Assimilation Pathway

scholarly journals Phosphoethanolamine Modification of Lipid A in Colistin-Resistant Variants of Acinetobacter baumannii Mediated by the pmrAB Two-Component Regulatory System

2011 ◽  
Vol 55 (7) ◽  
pp. 3370-3379 ◽  
Author(s):  
Alejandro Beceiro ◽  
Enrique Llobet ◽  
Jesús Aranda ◽  
José Antonio Bengoechea ◽  
Michel Doumith ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Lipid A ◽  
Gene Knockout ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Strain Atcc ◽  
Colistin Resistance ◽  
Content Type ◽  
Knockout Mutants

ABSTRACTColistin resistance is rare inAcinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient.pmrABsequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression ofpmrA(4- to 13-fold),pmrB(2- to 7-fold), andpmrC(1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg2+induction conditions.pmrBgene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression ofpmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance inA. baumanniirequires distinct genetic events, including (i) at least one point mutation inpmrB, (ii) upregulation ofpmrAB, and (iii) expression ofpmrC, which lead to addition of phosphoethanolamine to lipid A.

scholarly journals An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

2016 ◽  
Vol 82 (12) ◽  
pp. 3481-3492 ◽  
Author(s):  
Tina Netzker ◽  
Volker Schroeckh ◽  
Matthew A. Gregory ◽  
Michal Flak ◽  
Mario K. C. Krespach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Deletion ◽  
Gene Knockout ◽  
Plasmid Transfer ◽  
Gene Deletions ◽  
Content Type ◽  
Efficient Generation ◽  
Knockout Mutants ◽  
Starter Unit ◽  
Targeted Gene Knockout

ABSTRACTStreptomyces iranensisHM 35 is an alternative rapamycin producer toStreptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation inS. rapamycinicus.The approach depends upon efficient conjugational plasmid transfer fromEscherichia colitoStreptomyces, and the failure of this step has frustrated its application toStreptomyces iranensisHM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions inS. iranensisHM 35. Among them wererapK, which generates the starter unit for rapamycin biosynthesis, andhutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants ofStreptomycesspecies that are genetically difficult to manipulate.IMPORTANCEThe developed protocol of conjugational plasmid transfer fromEscherichia colitoStreptomyces iranensismay allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable,Streptomycesspecies.

scholarly journals Coordinated and Distinct Functions of Velvet Proteins in Fusarium verticillioides

2014 ◽  
Vol 13 (7) ◽  
pp. 909-918 ◽  
Author(s):  
Nan Lan ◽  
Hanxing Zhang ◽  
Chengcheng Hu ◽  
Wenzhao Wang ◽  
Ana M. Calvo ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Affinity Purification ◽  
Fusarium Verticillioides ◽  
Toxin Production ◽  
Transcriptional Analysis ◽  
Phenotypic Characterization ◽  
Morphological Development ◽  
Content Type ◽  
Knockout Mutants ◽  
Fungal Kingdom

ABSTRACTVelvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogenFusarium verticillioides, previous studies showed that the velvet proteinF. verticillioidesVE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteinsF. verticillioidesVelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene,F. verticillioidesCAT2(FvCAT2). Deletion ofFvCAT2resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom.

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