scholarly journals An inside look at a biofilm: Pseudomonas aeruginosa flagella biotracking

2021 ◽  
Vol 7 (24) ◽  
pp. eabg8581
Author(s):  
Eden Ozer ◽  
Karin Yaniv ◽  
Einat Chetrit ◽  
Anastasya Boyarski ◽  
Michael M. Meijler ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Life Cycle ◽  
Opportunistic Pathogen ◽  
Model Organisms ◽  
Wild Type ◽  
Bacterial Flagella ◽  
Knockout Strain ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Genetic Code Expansion

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

scholarly journals An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

2020 ◽  
Author(s):  
Eden Ozer ◽  
Karin Yaniv ◽  
Einat Chetrit ◽  
Anastasya Boyarski ◽  
Michael M. Meijler ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Unnatural Amino Acid ◽  
Model Organisms ◽  
Wild Type ◽  
Bacterial Flagella ◽  
Knockout Strain ◽  
Biofilm Structure

AbstractThe opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a “flagella knockout” strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

scholarly journals Adaptation of Aerobically Growing Pseudomonas aeruginosa to Copper Starvation

2008 ◽  
Vol 190 (20) ◽  
pp. 6706-6717 ◽  
Author(s):  
Emanuela Frangipani ◽  
Vera I. Slaveykova ◽  
Cornelia Reimmann ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Cellular Respiration ◽  
Wild Type ◽  
Respiratory Enzymes ◽  
Terminal Oxidases ◽  
Copper Status ◽  
In The Wild ◽  
Quadruple Mutant ◽  
Heme Copper Oxidases

ABSTRACT Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA′-′lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.

scholarly journals Pyocyanin Alters Redox Homeostasis and Carbon Flux through Central Metabolic Pathways in Pseudomonas aeruginosa PA14

2007 ◽  
Vol 189 (17) ◽  
pp. 6372-6381 ◽  
Author(s):  
Alexa Price-Whelan ◽  
Lars E. P. Dietrich ◽  
Dianne K. Newman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Metabolic Pathways ◽  
Carbon Flux ◽  
Redox Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Physiological Relevance ◽  
In The Wild ◽  
Phenazine Production

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD+ in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.

scholarly journals Serine 229 Balances the Hepatitis C Virus Nonstructural Protein NS5A between Hypo- and Hyperphosphorylated States

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
Chia-Ni Tsai ◽  
Ting-Chun Pan ◽  
Cho-Han Chiang ◽  
Chun-Chiao Yu ◽  
Shih-Han Su ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Feedback Regulation ◽  
Wild Type ◽  
Serine Residue ◽  
Large Structures ◽  
Phosphorylation Level ◽  
In The Wild

ABSTRACT The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle. IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

scholarly journals Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1407-1415 ◽  
Author(s):  
James E. A. Zlosnik ◽  
Gholam Reza Tavankar ◽  
Jacob G. Bundy ◽  
Dimitris Mossialos ◽  
Ronan O'Toole ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Toxic Agent ◽  
Stationary Phase Culture ◽  
Prolonged Incubation ◽  
Mutant Strains ◽  
Electron Sink ◽  
Culture Supernatants

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

scholarly journals TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

2014 ◽  
Vol 83 (3) ◽  
pp. 863-875 ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Daniel A. Powell ◽  
Angela T. Nguyen ◽  
Maura O'Neill ◽  
Louise Djapgne ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Homeostasis ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Genes Encoding ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Heme Regulation ◽  
Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

scholarly journals Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa

2001 ◽  
Vol 183 (18) ◽  
pp. 5213-5222 ◽  
Author(s):  
Thilo Köhler ◽  
Christian van Delden ◽  
Lasta Kocjancic Curty ◽  
Mehri Michea Hamzehpour ◽  
Jean-Claude Pechere
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Sensing Systems ◽  
In The Wild ◽  
Type Mutant ◽  
Regulator Genes

ABSTRACT Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase genelasB and the rhamnosyltransferase genesrhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL andPseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.

scholarly journals Embryonic geometry underlies phenotypic variation in decanalized conditions

2019 ◽  
Author(s):  
A. Huang ◽  
T. E. Saunders
Keyword(s):  
Phenotypic Variation ◽  
Regulatory Network ◽  
Physical Space ◽  
Model Organisms ◽  
Wild Type ◽  
Environmental Perturbations ◽  
Key Factor ◽  
In The Wild ◽  
Gene Regulatory ◽  
Anterior Posterior

AbstractDuring development, many mutations cause increased variation in phenotypic outcomes, a phenomenon termed decanalization. Such variations can often be attributed to genetic and environmental perturbations. However, phenotypic discordance remains even in isogenic model organisms raised in homogeneous environments. To understand the mechanisms underlying phenotypic variation, we used as a model the highly precise anterior-posterior (AP) patterning of the early Drosophila embryo. We decanalized the system by depleting the maternal bcd product and found that in contrast to the highly scaled patterning in the wild-type, the segmentation gene boundaries shift away from the scaled positions according to the total embryonic length. Embryonic geometry is hence a key factor predetermining patterning outcomes in such decanalized conditions. Embryonic geometry was also found to predict individual patterning outcomes under bcd overexpression, another decanalizing condition. Further analysis of the gene regulatory network acting downstream of the morphogen identified vulnerable points in the networks due to limitations in the available physical space.

scholarly journals Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Ian T. Hill ◽  
Thomas Tallo ◽  
Matthew J. Dorman ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Toxic Effects ◽  
Growth Defect ◽  
Wild Type ◽  
Rna Chaperone ◽  
Small Protein ◽  
Content Type ◽  
Transcription Regulator ◽  
Mutant Cells

ABSTRACT Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

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