Catabolite Repression Control of Pyocyanin Biosynthesis at an Intersection of Primary and Secondary Metabolism in Pseudomonas aeruginosa

Huang, Jiaofang; Sonnleitner, Elisabeth; Ren, Bin; Xu, Yuquan; Haas, Dieter

doi:10.1128/aem.00026-12

Cited by 40 publications

(58 citation statements)

References 32 publications

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“…Because phenazines alter the metabolic flux of P. aeruginosa (7,20), we reasoned that intermediates of central metabolism might also serve as electron donors, and so we also tested pyruvate, citrate, isocitrate, ␣-ketoglutarate, succinate, and malate. We prepared cell lysate from late-exponential phase cultures grown on succinate, a preferred carbon source of P. aeruginosa (33). Because phenazines induce a transcriptional response in P. aeruginosa (34), and so may regulate the activity of their endogenous reductases, we harvested the cultures several hours after the onset of phenazine production as indicated by the blue color of pyocyanin.…”

Section: Resultsmentioning

confidence: 99%

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Glasser

Wang

Hoy

et al. 2017

Journal of Biological Chemistry

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Edited by Joseph JezPhenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and hemecontaining proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and ␣-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or ␣-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD ؉ (1.45 Å), and bound to NADH (1.79 Å).In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD ؉ in LpdG and other enzymes, achieving the same end by a different mechanism.

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Section: Resultsmentioning

confidence: 99%

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Glasser

Wang

Hoy

et al. 2017

Journal of Biological Chemistry

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“…As noted before, large amounts of succinate (ϳ6 mM) were produced during growth of KRP1 on glucose, and the reconsumption of this intermediate correlates in time with the highest observed electrochemical activity in the glucose experiments. For P. aeruginosa (strain PAO1), it was shown before that the highly preferred carbon substrate succinate suppresses PYO synthesis (and to a lesser extent also PCA synthesis) through catabolite repression via the Crc protein (48). It should be investigated whether a change in this regulatory path is responsible for the strong production of PCA and electric current in strain KRP1.…”

Section: Discussionmentioning

confidence: 99%

Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa

Bosire

Blank

Rosenbaum

2016

Appl Environ Microbiol

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Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa. We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 A cm ؊2 with ϳ150 g ml ؊1 phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions. IMPORTANCEMicrobial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial community to access a solid electrode as an alternative electron acceptor. To better understand the ecological relationships between mediator producers and mediator utilizers, we here present a comparison of the phenazine-dependent electroactivities of three Pseudomonas strains. This work forms the foundation for more complex coculture investigations of mediated electron transfer in microbial fuel cells. Bioelectrochemical systems (BES), including their most important variant, the microbial fuel cell (MFC), are rapidly developing and promising technologies for renewable energy production and wastewater treatment, among other applications (1, 2). The MFC technology aims at generating electrical current through extracellular transfer of electrons, which microorganisms liberate from organic substrates. Microorganisms oxidize organic compounds, and the electrons from the intracellular electron transport chains are transferred to an external electron acceptor (i.e., an anode poised at a suitable potential) (3). One of the challenges facing MFC performanc...

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“…9 Crc is involved in several Pseudomonas species in catabolite repression of the branched-chain keto acid dehydrogenase 23 and of alkane degradation, 7 as well as of a number of enzymes involved in aromatic compound degradation. 24 In addition to modulating metabolism, in P. aeruginosa, Crc influences susceptibility to antibiotics, expression of Type III secretion, expression of quorum sensing-regulated virulence factors, such as pyocyanin 25 and potentially other virulence functions. 26 Sugars such as glucose, sucrose and fructose are known to be inducers of the P. syringae TTSS genes, whereas tricarboxylic acids (TCA) intermediates can suppress T3SS in vitro.…”

Section: Discussionmentioning

confidence: 99%

CrcZ and CrcX regulate carbon source utilization inPseudomonas syringaepathovartomatostrain DC3000

et al. 2013

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Catabolite Repression Control of Pyocyanin Biosynthesis at an Intersection of Primary and Secondary Metabolism in Pseudomonas aeruginosa

Cited by 40 publications

References 32 publications

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa

CrcZ and CrcX regulate carbon source utilization inPseudomonas syringaepathovartomatostrain DC3000

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