Microbial Production of Phenazines

Ingram, J. M.; Blackwood, A. C.

doi:10.1016/s0065-2164(08)70406-4

Cited by 39 publications

(28 citation statements)

References 36 publications

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“…We began by focusing on three endogenous phenazinespyocyanin (PYO), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-OHPHZ)-that are known to be excreted by PA14 during stationary-phase growth cycle in laboratory cultures (9,17). We harvested cells from cultures grown aerobically on Luria-Bertani (LB; Fisher Scientific) medium at 37°C and concentrated and resuspended them in anaerobic MOPS medium at 10 9 CFU/ml.…”

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Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

2010

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Phenazines have long been recognized for their redox properties. While most attention concerning their redox activity has focused on their role in generating reactive oxygen species in the context of infection (13,14,19,22), as early as 1931, Friedheim hypothesized that phenazine reduction might benefit producer cells as an alternative respiratory pigment (12). Several years ago, our group suggested that the context in which this might be most important would be in biofilms, where cell densities are high and access to oxidants is limited (16,31); consistent with this, we recently showed that mutants unable to produce phenazines are defective in community development (8). While this phenotype is likely due to many factors, including a signaling function for phenazines in later stages of growth (9), given the 1931 hypothesis by Friedheim (12) and our related recent work demonstrating that phenazines control redox homeostasis in Pseudomonas aeruginosa (30), we reasoned that phenazines might contribute to the survival of cells experiencing oxidant limitation.As a first step toward testing this, we investigated the effect of redox-active small molecules on anaerobic survival of P. aeruginosa PA14 in stationary-phase planktonic cultures. We justified beginning with planktonic cultures rather than biofilms based on previous studies which have suggested that cells in stationary-phase planktonic culture physiologically resemble cells in established biofilms (15,35,38). Moreover, by working with cells in planktonic cultures, we could build on voltammetric methods that had previously been used to determine how metabolism changes in Escherichia coli in the presence of the synthetic redox-shuttle ferricyanide (36). Similar voltammetric approaches have also been used to study how phenazines (32, 33) and structurally related flavins (23) mediate power generation by microbial fuel cells.We assembled bulk electrolysis-based glass bioreactors housed within an O 2 -and H 2 -free glove box (MBraun) and controlled by a multichannel potentiostat (series G 300; Gamry) outside the glove box. Each bioreactor held a graphite rod working electrode (Alfa Aesar) with an operating surface area of 6 cm 2 , a Ag/AgCl reference electrode (RE-5B; BASi) with a constant potential of ϩ207 mV versus that of the normal hydrogen electrode (NHE), and about 100 ml MOPS (morpholinepropanesulfonic acid) culture medium (100 mM MOPS at pH 7.2, 93 mM NH 4 Cl, 43 mM NaCl, 2.2 mM KH 2 PO 4 , 1 mM MgSO 4 , 5.0 M FeCl 3 ) (modified from reference 29). The bioreactor was joined by a fritted glass junction to a small side chamber, in which a Pt counter electrode made from Pt mesh (Alfa Aesar) soldered to a copper wire completed the circuit. In order to selectively examine different redox-active small molecules, we used the phenazine-null mutant of PA14 (⌬phz) which is deleted in its two phenazine biosynthetic operons (9).We began by focusing on three endogenous phenazinespyocyanin (PYO), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-OHPHZ)-that are ...

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Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

2010

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“…1) is the characteristic blue-green phenazine pigment produced by P. aeruginosa. Pyocyanin is the most thoroughly studied of the phenazine pigments (13,33). Phenazines are classified as secondary metabolites, i.e., compounds formed during the stationary phase and often having antibiotic properties.…”

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Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications

et al. 1990

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We have found that Pseudomonas aeruginosa has two anthranilate synthase enzymes; they are homologous, and both have conventional ot and p subunits (9b). The two anthranilate synthases have different functions. One participates in tryptophan synthesis; its genes have been designated trpE and trpG (9b). These genes are quite similar to those of Pseudomonas putida (9a). In both pseudomonads, trpG, which encodes the p subunit, is cotranscribed with trpD and trpC in a three-gene operon.The second anthranilate synthase, previously misidentified as the product of a trp gene pair, was obtained on a R-prime plasmid by mating P. aeruginosa PAC174 carrying R68.44 with Escherichia coli W3110 tna AtrpE5 (8). This R-prime plasmid complements only those E. coli auxotrophs blocked in anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. Enzyme assays indicated that both the a and P subunits of anthranilate synthase were produced. The anthranilate synthase-encoding segment of the R-prime plasmid was subcloned into pBR322 (8). DNA sequencing of one of these subclones, pIA14, showed that the genes for the a and P anthranilate synthase subunits were indeed present on the cloned DNA. These genes are adjacent, and their coding sequences overlap by 23 base pairs * Corresponding author. t Present address:

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“…The structural similarities between redox-active antibiotics and known electron shuttles not only explain why they all utilize TolC-linked efflux systems but also indicate that they may have similar functions (9). For example, pyocyanin, a phenazine blue pigment that is typically considered to be an antibiotic due to the generation of toxic intermediates during its oxidation (7,8), may also function as an electron shuttle during aerobic growth of Pseudomonas aeruginosa (6,10). Indeed, the same underlying chemistry that makes redox-active antibiotics toxic to some cells (11) may in some contexts (such as respiratory growth with limiting terminal electron acceptors) be beneficial to other cells.…”

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Protective Role of tolC in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate

2002

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Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer. Here we provide genetic evidence that AQDS enters Shewanella oneidensis strain MR-1 and causes cell death if it accumulates past a critical concentration. A tolC homolog protects the cell from toxicity by mediating the efflux of AQDS. Electron transfer to AQDS appears to be independent of the tolC pathway, however, and requires the outer membrane protein encoded by mtrB. We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics.

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Microbial Production of Phenazines

Cited by 39 publications

References 36 publications

Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications

Protective Role of tolC in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate

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