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Figure 1: Molecular structure of phenazine.

Phenazines are water-soluble secondary metabolites secreted by some bacterial species. Almost all their properties can be attributed to their ability to undergo redox transformations. Some soil-born bacteria commonly produce phenazine-1-carboxylic acid (PCA)) against fungal root disease. (Niknejad Kazempour, 2009) Bacteria are also able to use this substance as a mediator for electron shuttling, which leads to an efficiency gain if a phenazine-producing strain is present in a MFC.
Problems arise with the different phenazine producing host strains. Most of them are classified as pathogens and therefore unsuitable for application in our lab and MFC. Hence, all experiments carried out in this section were soluble done with Pseudomonas fluorescences which is classified as risk group 1.
Initially PCA overproduction seemed to be a promising approach to achieve higher electron transfers. Because of the extremely complicated cloning procedure as well as the good advice of the consulted MFC expert Dr. Falk Harnisch, we abandoned this sub-project in an early stage and searched for alternatives. We concentrated on an overexpression of riboflavin, which serves as an electrone shuttle itself and of the glycerol dehydrohenase gldA, which increases NADH concentration. (see sections Riboflavin and gldA on our Wiki for more information). Still, quite a lot of work was invested into investigation of phenazine functions and synthesis in bacteria, of so we present a short theoretical overview of this topic. The only entry in the labjournal on phenazine is found here in Week 13.


Phenazines comprise a large group of heterocyclic nitrogen-containing substances, having a tricyclic ring in its core. They are substituted at different points around their rings, which alters water solubility and other properties. After discovery of naturally occurring phenazines, more than 6000 were chemically synthesized. (Mavrodi, 2006). Neutral red, a well-known pH-indicator, also belongs to this class.

Phenazine derivatives were one of the first secondary bacterial metabolites that were consequently studied since their discovery in 1859. The first substance of this family, a blue pigment of Pseudomonas aeruginosa, was called «pyocyanine» (short PYO). It has antimicrobial properties that raised further interest in these substances. Phenazines are produced by many bacterial species, but they are mostly studied in representatives of fluorescent pseudomonads. A single bacterial strain usually produces two or more species-specific phenazines, except Pseudomonas fluorescens 2-79 that is known to produce only PCA, a broad-spectrum antibiotic, which is a casual agent active against a variety of root pathogens (Mavrodi et al., 2001).

Biological phenazines are known for their broad-spectrum of antibiotical, antiparasaticand even anti-malaria properties (Handelsmann et al., 1996). Consequently, it was shown that almost all properties of phenazines could be attributed to their ability to undergo redox transformations (Muller, 1995). This discovery brought up the idea, that these molecules can be used as an electron shuttle in a MFC. Indeed, it was shown that external addition of phenazines, both PCA and PCN (phenazine-1-carboxamide) alike, causes a significant raise of current flow (The Hai Pham, 2008). Surprisingly, a mixed culture of phenazine-secreting and phenazine non-secreting strains showed similar results, proving that phenazine can be used for redox cycling not only by its producer (Hernandez et al. 2004).

Many strains of Pseudomonas fluorescens, common soil inhabitants, are known to produce the phenazine compound PCA. The phenazine synthesis pathway was studied in detail. The seven-gene cluster phzABCDEFG, 8505 bp long, was firstly identified in Pseudomonas fluorescens 2-79.

Research on phenazine diversity and genetic evolution, (Mavrodi, 2010) stated, that many species of fluorescent pseudomonas have the corresponding genes and are able to produce phenazines, so we expected that orthologs are found in the Pseudomonas fluorescens 50090 that we obtained from DSMZ.

Figure 2: Comparison of sequences between enzymes in phenazine cluster and other proteins with known function, according to research of MacDonald et al. 2001.

Genetic Approach

Figure 3: Phenazine synthesis cluster, comprising an autoinducer synthase (phzI) gene, positive regulator protein (phzR) and pathway-coding phzABCDEFG genes.

The coding cluster revealed seven forbidden restriction sites, making a cloning in accordance with the iGEM-rules a steep challenge. Unfortunately, amplification of the cluster was never successful. We varied different parameters and tried different enzymes, but we always saw a false priming and multiple amplificates. So we decided to look for applicable alternatives. Riboflavin, a natural mediator seemed an alternative of choice and our decision was rewarded by successful results.


There are no practical results in this section. The project was set aside after unsuccessful attempts to amplify the phenazine-coding fragment from Pseudomonas fluorescens sp. The only entry in the labjournal on phenazine is found here in Week 13.


  • Mavrodi D.V, Tomashow L.S. (2006) Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annual review of Phytopathology 44:417– 445
  • Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS (2001) Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183:6454–6465
  • Muller M. Scavenging of neutrophil-derived superoxide anion by 1-hydroxyphenazine, a phenazine derivative associated with chronic Pseudomonas aeruginosa infection: relevance to cystic fibrosis. (1995) Biochim Biophys Acta: 1272(3):185-9.
  • McDonald M, Mavrodi DV, Thomashow LS, Floss HG. (2001). Phenazine biosynthesis in Pseudomonas fluorescens: branchpoint from the primary shikimate biosynthetic pathway and role of phenazine-1,6-dicarboxylic acid. J. Am. Chem. Soc. 123:9459–60