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Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide (1999)

Hassett, Daniel J. ; Ma, Ju-Fang ; Elkins, James G. ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 34 (1999), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-l-homoserine lactone) and PAI-2 (N-butyryl-l-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. β-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01672.x

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GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes (2002)

Ochsner, Urs A. ; Wilderman, Paula J. ; Vasil, Adriana I. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 45 (2002), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip® to analyse the global gene expression profiles in response to iron. In iron-starved cells, the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip® data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease, PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron-controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron-regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low-iron conditions, expression of 26 genes or operons was reduced in a ΔpvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip® proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2002.03084.x

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3

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Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis (2001)

Rahim, Rahim ; Ochsner, Urs A. ; Olvera, Clarita ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 40 (2001), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso-active glycolipids containing one (mono-rhamnolipid) or two (di-rhamnolipid) l-rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono-rhamnolipid from dTDP-l-rhamnose and β-hydroxydecanoyl-β-hydroxydecanoate, whereas di-rhamnolipid is produced from mono-rhamnolipid and dTDP-l-rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di-rhamnolipid (l-rhamnose-l-rhamnose-β-hydroxydecanoyl-β-hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of l-rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII-2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di-rhamnolipid, whereas mono-rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1-rhlA) was incapable of producing both mono- and di-rhamnolipid. Complementation of mutant RTII-2 with plasmid pRTII-26 containing rhlC restored the level of di-rhamnolipid production in the recombinant to a level similar to that of the wild-type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A σ54-type promoter for the PA1131–rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131–rhlC operon was dependent on the P. aeruginosa rhl quorum-sensing system, and a well-conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di-rhamnolipid.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2001.02420.x

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4

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Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor (1996)

Ochsner, Urs A. ; Johnson, Zaiga ; Lamont, Iain L. ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 21 (1996), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron-limiting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. The expression of both toxA and regAB is repressed under iron-replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur-regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin. In a ΔpvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild-type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Neither toxA nor regAB mRNA was detectable in a ΔpvdS mutant. Overexpression of pvdS from the tac promoter on a plasmid resulted in a high-level and iron-independent production of ETA in wild-type PAO1, in the ΔpvdS strain, but not in a ΔregA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the Ptac–pvdS gene was monitored in cells grown in high-iron medium. While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mechanism besides the Fur–PvdS system is involved in iron regulation of ETA production.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1996.481425.x

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5

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Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production (1996)

Hamood, Abdul N. ; Colmer, Jane A. ; Ochsner, Urs A. ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 21 (1996), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron-deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four- to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb KpnI–BglII fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino-terminus region of PtxR contains a putative helix-turn-helix DNA-binding motif. Specific ptxR-deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA–lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1996.6251337.x

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6

Electronic Resource

The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence (1999)

Vasil, Michael L. ; Ochsner, Urs A.

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 34 (1999), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator — Fur) and a Fur-regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01586.x

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7

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Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes (1991)

Ochsner, Urs A. ; Glumoff, Virpi ; Kälin, Markus ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Yeast 7 (1991), S. 513-524

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ISSN: 0749-503X

Keywords: Trichosporon cutaneum ; auxotrophic mutants ; UV-mutagenesis ; transformation of spheroplasts ; sib-selection ; integration ; Life and Medical Sciences ; Genetics

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology

Notes: A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully in spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per μg of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementing cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed. T. cutaneum spheroplasts with an efficiency of 50-200 colonies per μg of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.

Additional Material: 7 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/yea.320070511

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