World J Microbiol Biotechnol (2014) 30:1159–1168

DOI 10.1007/s11274-013-1552-5

REVIEW

Pyocyanin: production, applications, challenges and new insights

Sheeba Jayaseelan • Damotharan Ramaswamy •

Selvakumar Dharmaraj

Received: 22 May 2013 / Accepted: 31 October 2013 / Published online: 9 November 2013
Ó Springer Science+Business Media Dordrecht 2013

Abstract Pseudomonas aeruginosa is an opportunistic, Introduction

Gram-negative bacterium and is one of the most com-
mercially and biotechnologically valuable microorganisms. Pseudomonas aeruginosa is a member of the Gamma
Strains of P. aeruginosa secrete a variety of redox-active Proteobacteria class of bacteria. Based on the revisionist
phenazine compounds, the most well studied being pyo- taxonomy done by analysis of conserved macromolecules
cyanin. Pyocyanin is responsible for the blue-green colour (e.g. 16S ribosomal RNA), the genus Pseudomonas was
characteristic of Pseudomonas spp. It is considered both as included in the bacterial family Pseudomonadaceae
a virulence factor and a quorum sensing signalling mole- (Glazebrook et al. 1978). Other members in the genus
cule for P. aeruginosa. Pyocyanin is an electrochemically include P. alcaligenes, P. anguilliseptica, P. citronellolis,
active metabolite, involved in a variety of significant bio- P. flavescens, P. jinjuensis, P. mendocina, P. nitroredu-
logical activities including gene expression, maintaining cens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans
fitness of bacterial cells and biofilm formation. It is also and P. straminae.
recognised as an electron shuttle for bacterial respiration Pseudomonas aeruginosa is a Gram-negative, aerobic rod
and as an antibacterial and antifungal agent. This review shaped bacterium measuring 0.5–0.8 lm wide and
summarises recent advances of pyocyanin production from 1.5–3.0 lm long. Almost all strains are motile by means of a
P. aeruginosa with special attention to antagonistic prop- single polar flagellum (Palleroni 2005; Moore et al. 2006). It
erty and bio-control activity. The review also covers the thrives not only in normal atmospheric conditions but also in
challenges and new insights into pyocyanin from P. hypoxic atmospheres, and has colonised many natural and
aeruginosa. artificial surroundings (Green et al. 1974; Migula 1984;
Suthar et al. 2009). Its habitat is thus widespread and it is
Keywords Antimicrobial effect
Bio-control agent
found in soil, water and many other environments.
Pigment production
Pyocyanin
Pseudomonas Pseudomonas aeruginosa attracts attention because of
aeruginosa its colour and pigment production. One of the most rec-
ognisable signs of an unknown colony being P. aeruginosa
is the characteristic fruity, grape-like odour derived from
the production of 2-aminoacetophenone by the organism.
On sheep blood agar plates, colonies of P. aeruginosa often
display beta-haemolysis and a greenish metallic sheen due
to its pigment production. No other species of Gram-neg-
S. Jayaseelan
S. Dharmaraj (&)
Dr. Sir A. L. Mudaliar Vocational Arts and Science College, ative non-fermenting bacteria produce pyocyanin, making
Vengal 601103, Tamil Nadu, India its presence helpful in identifying the organism. Pigments
e-mail: [email protected] secreted by P. aeruginosa include pyocyanin (blue-green),
pyoverdin (yellow, green and fluorescent), pyomelanin
S. Jayaseelan
D. Ramaswamy
Department of Plant Biology and Biotechnology, Presidency (light-brown) and pyorubrin (red-brown) (Reyes et al.
College, Chennai 600005, Tamil Nadu, India 1981; Meyer 2000).

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Nearly 90–95 % of all isolates of P. aeruginosa produce contained glycerol. The addition of nalidixic acid to this
the pigment pyocyanin, which is deep blue in colour and medium, with decreased concentration of cetrimide,
referred to as ‘‘blue pus’’ (from pyocyaneus) (Fordos 1863; inhibited contaminating flora and thus allowed for better
Gessard 1984; Ran et al. 2003). Pyocyanin is produced recovery of P. aeruginosa with a concomitant increase in
abundantly in media with low iron content and plays an pyocyanin production (Brown and Lowbury 1965; Lilly
important role in iron metabolism. Pyocyanin appears to and Lowbury 1972).
participate in a reduction mechanism which is capable of Recently there was a report on production of pyocyanin
reducing and releasing the iron from transferrin. Iron is a from hundred strains of P. aeruginosa isolated from clin-
crucial requirement for the growth of P. aeruginosa (Cox ical samples using various modified medium (Sheeba
1986). Pyocyanin pigment is a phenazine which is a 2007). Seven liquid media [Frank’s medium (FM) ? 1 %
nitrogen-containing heterocyclic compound. It is a redox yeast extract, FM ? 0.5 % tryptone, FM ? 0.1 % KNO3,
active secondary metabolite and is soluble in chloroform. FM ? 1 % bacto-peptone, FM ? 0.1 % glucose, peptone,
This metabolite, 1-hydroxy-N-methyl phenazine, contrib- nutrient broth] and six solid media (Tryptone glucose yeast
utes to bacterium survival. Little is known about the two agar, Seller’s differential medium, Nutrient agar, FNA
enzymes designated PhzM and PhzS that function in the medium, Cetrimide agar and Pseudomonas agar) were used
synthesis of pyocyanin from the precursor phenazine-1- for pigment production. Frank’s medium [FM] ? 1 %
carboxylic acid. Phenazine compounds produced from the yeast extract showed highest production of pyocyanin than
rhizosphere of plants contribute to the biological activity of the other liquid media. Among the six solid medium used,
P. aeruginosa against Fusarium (wilt of chickpea) and cetrimide agar showed maximal production of pyocyanin.
Pythium (damping-off of bean). The production of bacterial pigment makes its identifica-
In this review, production, purification, antagonistic tion more rapid from clinical samples due to the antibiotic
property, bio-control activity, challenges and new insights activity of the pigment.
of pyocyanin from P. aeruginosa are explained in detail. There are some factors that influence pigment produc-
tion when using various media. Production of pyocyanin
from Pseudomonas wild type and mutant strains was
Pyocyanin production and factors that influence investigated, and from the study it was concluded that
production phosphate depletion in the culture medium enhances pig-
ment production (Ingledew and Campbell 1969; Byng et al.
Previously, pyocyanin production was carried out using 1979; Porter 2009). In another report, there is evidence that
King’s Medium (20 g peptone, 1.4 g magnesium chloride, depletion of iron in the medium favours pyocyanin pro-
10 g potassium sulphate, 15 g agar in 1 l of distilled water, duction (Cox 1986). Depending on the cell density, the
pH 7.0 ± 0.2, 25 °C) as described by King et al. in 1954. organism produces pigment and when there is a depletion
Based on King’s Medium, Pseudomonas agar medium was of some nutrients, there is more production. It has been
formulated which is recommended for pyocyanin prodcu- reported that a satisfactory yield of pyocyanin depends
tion by Pseudomonas species. This medium enhances the mainly on the composition of the media (Chang and
elaboration of pyocyanin but inhibits the formation of other Blackwood 1969; Reyes et al. 1981).
pigments. There are many reports which determined the
cultural characteristics and conditions which favour the
production of pyocyanin (Burton et al. 1948; Karpagam Pigment extraction and purification
et al. 2013; Sudhakar et al. 2013). Combination of amino
acids, notably alanine and leucine, in the culture medium Formerly, liquid glycerol–peptone–phosphate medium was
resulted in a more effective pigment production. There used for enhancing pyocyanin production from P. aeru-
were several works on designing the medium for pigment ginosa. The pyocyanin pigment which is diffusible into the
production. Media formulations that produce optimal levels medium can be solvent-extracted by chloroform. The
of pyocyanin contain glycerol, alanine, sulphur, and iron. extracts were purified using a column of aluminium oxide
Presence of alanine and glycerol as joint substrates was with elution solvent mixture of chloroform-ethanol and
highly effective and acted as a precursor for pyocyanin crystallised (El-Samerraie et al. 1997). Later in another
production. This joint substrate medium is the Frank and report, the pigment was isolated by various biochemical
De Moss medium, recommended for the rapid diagnosis of profiling and the peptide was purified by ammonium sul-
P. aeruginosa and demonstration of pyocyanin (Frank and phate precipitation, gel filtration and ion-exchange chro-
De Moss 1958; Wahba and Darrel 1965; Subramaniam and matography (Fontoura et al. 2009). O’Malley et al. (2003)
Shriniwas 1985). A modified formulation of King’s Med- extracted pyocyanin using 10 mM hydrochloride followed
ium for pyocyanin production was developed which by neutral water instead of chloroform and this was

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confirmed by high pressure liquid chromatography phenazines. In the first step, it is catalyzed by the enzyme
(HPLC). There is a report on extraction of the pyocyanin PhzM, an S-adenosylmethionine (SAM) dependent meth-
pigment using nine different solvents and among them, yltransferase, in which PCA is converted to 5-methylphe-
chloroform showed the highest extractability of the pig- nazine-1-carboxylic acid betaine by transfer of a methyl
ment. The purity of the extracted pigment was checked by group to the nitrogen atom of phenazine-ring moiety. The
thin layer chromatography. In this study, pyocyanin was second step is catalyzed by the enzyme PhzS, a FAD-
extracted with 3 ml of chloroform and re-extracted with dependent monooxygenase, and involves the hydroxylative
1 ml of 0.2 N HCl (Hassanein et al. 2009; Raoof and Latif decarboxylation of 5-methylphenazine-1-carboxylic acid
2010). betaine to pyocyanin (Parsons et al. 2007).
Saosoong et al. (2009) reported pyocyanin production
from P. aeruginosa. The pigment was purified by passing
the supernatant in Amberlite XAD-4 resin column and Anti-bacterial activity of pyocyanin
concentrating the pigment with a silica gel column. It was
re-chromatographed by HPLC and purity was checked by Pyocyanin is a good example of a secondary metabolite,
both UV/visible and IR spectroscopy. Porter (2009) also which has antibiotic activities and also able to co-ordinate
reported the production of pyocyanin from P. aeruginosa the response of microbial communities to changes in the
and it was extracted using chloroform. The pigment was environment. Little attempts have been made to determine
found to be produced by the inner part of the cells which the relation between pyocyanin and its inhibitory action.
was lysed by addition of chloroform. The purity was con- Anti-bacterial activity of pyocyanin has been reported
firmed by passing the aqueous solution into a Sephadex since the year 1940 (Waksman and Woodruff 1940). The
G-10 column. pigment inhibited the growth of Escherichia coli and was
named as Colicin. The protein fraction was liberated upon
lysis of bacteria which exhibited the properties of pyocy-
Pyocyanin structure and involvement of genes anin (Young 1947). The purified form of pyocyanin
showed antibacterial activity and depending on the con-
The pigment pyocyanin is composed of 2 subunits of N- centration of pyocyanin, the bactericidal effect varied
methyl-1-hydroxyphenazine. Pyocyanin has been crystal- (Baron and Rowe 1981). The mechanism by which pyo-
lized in pure form and classified as a phenazine-type of cyanin inhibits bacterial growth was investigated and it was
molecule (Norman et al. 2004). The biosynthesis of concluded that, pyocyanin interacts with the cell membrane
phenazine has been extensively studied in Pseudomonas respiratory chain resulting in the inability of the bacterial
(Hollstein and Marshall 1972; Hollstein and McCamey cells to perform their active metabolic transport process
1973; Herbert et al. 1974, 1976). Phenazine is a hetero- (Baron et al. 1989). Exposure of E. coli cultures to pyo-
cyclic compound that is produced naturally as the deep red cyanin causes depletion of oxygen supply to the cells,
5-methly-7-amino-1-carboxyphenazium betaine, which is produces H2O2 and also diverts the electron flow, causing
then converted to the lemon yellow coloured phenazine-1- toxicity to E. coli cells (Hassan and Fridorich 1980). Py-
carboxylic acid (PCA), and finally to the bright blue ocyanin negatively influences the active transport mecha-
1-hydroxy-5-methyl phenazine (pyocyanin) (Gohain et al. nism of several organisms (Baron et al. 1989).
2006). There are reports on anti-staphylococcal activity by P.
The shikimic acid pathway was found to be the primary aeruginosa. P. aeruginosa present in the sputum of cystic
metabolic pathway which branched into phenazine bio- fibrosis (CF) patients inhibited the growth of Staphylo-
synthesis. Shikimic acid pathway was the precursor for coccus aureus. The antagonistic effects were proved by
phenazine which gives a branching point for chorismic acid cross-streak test, well plate assay and growth of mixed
for the synthesis of phenazine (Hollstein and Marshall culture (Machan et al. 1990).
1972; Herbert et al. 1976; Millican 1962). In the synthesis The antibiotic action of pyocyanin from P. aeruginosa
of pyocyanin by P. aeruginosa seven genes have been isolated from the marine environment was studied by
identified, namely phz C, D, E, F, G, M and S. The phen- Angell et al. (2006). Marine P. aeruginosa were isolated
azine biosynthetic loci were found in all Pseudomonas from ocean sediments collected in Hawaiian Islands and
species (Mavrodi et al. 2001; Ahuja 2006; Gohain et al. screened for antimicrobial activity. A blue metabolite,
2006). Of these genes, phzM and phzS were the main genes pyocyanin was identified in P. aeruginosa Pup14B culture.
responsible for converting phenazine-1-carboxylic acid to Full characterisation of pyocyanin were done in the strain,
pyocyanin (Fig. 1). Two steps are suggested to be involved using X-ray analysis and 2D-NMR and corrections made in
1
in the synthesis of pyocyanin from PCA, which is the H and 13C-NMR assignments of the molecule misreported
common precursor for many different species-specific in the chemical literature and yeast transcriptome analysis.

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Fig. 1 Biosynthesis of pyocyanin from phenazine-1-carboxylic acid, a derivative of shikimic acid pathway

The transcriptional effect were consistent with the com- 1994). Substances like pyocyanin, pyrrolnitrin and
pound purported role as an inducer of oxidative stress and pseudomonic acid produced by P. aeruginosa showed
damage, and illustrates the overall potential of the method antibiotic actions in vivo on Candida species grown on
to reveal the primary biological/cellular effects of a natural Sabroud’s Dextrose Agar (Pal and Revathi 1998; Kaleli
product. et al. 2007). Signal mediated interactions between P.
Nearly 90–95 % of antimicrobial inhibitions of P. aeru- aeruginosa and C. albicans in CF patients was found. The
ginosa strains were due to production of the water soluble presence of N-acyl homoserine lactones (HSLS) produced
secondary metabolite pyocyanin. It showed antagonistic by P. aeruginosa affected the morphology of C. albicans.
activity against pathogenic bacteria like Salmonella paraty- In the same way C. albicans inhibited the swarming
phi, E. coli and Klebsiella pneumonia (Saha et al. 2008). Py- motility of P. aeruginosa. When P. aeruginosa was co-
ocyanin isolated from P. aeruginosa 4B strain showed cultured with C. albicans, the former synthesised large
antibiotic activities against various pathogens and food amounts of pyocyanin and even the growth of C. albicans
spoilage bacteria like Listeria monocytogens and Bacillus was inhibited (Hogan and Kolter 2002; Mc Alester et al.
cereus. The secondary metabolite along with various enzymes 2007; Gibson et al. 2009; Hassanein et al. 2009).
like haemolysin and hydrolytic enzymes played a key role for Sudhakar et al. (2013) reported the production of pyo-
their antimicrobial activities (Fontoura et al. 2009). cyanin from P. aeruginosa WS1 and its antagonistic
The development of simple and low cost micro-bioreactor activity against commonly encountered phytopathogens.
with high bio-processing for the production of pyocyanin The pyocyanin compound was extracted and purified using
from P. aeruginosa DSO-129 has been reported (Rahman gas chromatography–mass spectrometer (GC–MS) and
et al. 2009). The strain was inoculated in the bioreactor with a found to have a molecular weight of 210.23 kDa with an Rf
modified nutrient broth. The organisms produced the sec- value 11.94. The MIC of pyocyanin was further analysed
ondary metabolite pyocyanin in the medium which was fil- against phytopathogens and was found to be 64 lg/ml
tered, and demonstrated for antimicrobial effect on against Aspergillus flavus and Aspergillus fumigates, and
organisms like S. aureus, Staphylococcus epidermis, Bacil- 128 lg/ml against Candida species.
lus subtilis, Micrococcus luteus and Saccharomyces cerevi-
siae. There has been another report related to the antibiotic
activity of pyocyanin against different pathogens. The pig- Bio-control activity of pyocyanin
ment produced by the strain showed very effective activity
against organisms like E. coli, Acinetobacter, S. aureus and Biological control of plant diseases has been considered as
Streptococcus pneumonia (Sweden 2010). a valuable alternative method to manage various plant
diseases (Cook 1993). In plant pathology, the term bio-
control applies to the use of microbial antagonists to sup-
Anti-fungal activity of pyocyanin press plant diseases as well as the use of host specific
pathogens to control weed populations. The microbial
Pyocyanin isolated from P. aeruginosa also inhibited the agent that suppresses the pathogen is referred to as the
growth of fungi like Aspergillus fumigatus and Candida biological control agent (BCA). In the hostile and nutrient-
albicans isolated from the sputum of CF patients (Kerr limiting soil environment, plant roots are the places for
et al. 1999). There was a clinical evidence that pyocyanin both beneficial and deleterious soil borne microbes. Ben-
suppressed the growth of different species of C. albicans in eficial Pseudomonas species reach root surfaces chemo-
patients with lung infection. Reoccurring of C. albicans tactically by flagella motility and colonise them. It was
was noticed after the suppression of pyocyanin (Kerr found that Pseudomonas also promotes the plant growth.

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The root and rhizosphere offer an ecological niche (Bais tea, cotton and banana crops (Sunish kumar et al. 2004).
et al. 2006). The minimal inhibitory concentration of PCA was found to
The interaction of P. aeruginosa with plants as a ben- be 29 lg/ml for Sclerotium rolfsii NCM1084, which also
eficial association was found to be quite common. Recent inhibited the growth of phytopathogens such as Aspergillus
studies have provided a sight into this complex regulatory niger NCIM 1025, Fusarium oxysporum NCIM 1008, S.
network. P. aeruginosa, produces pyocyanin which is rolfsii NCIM1084 and Colletotricum falcatum (Rane et al.
present in the rhizosphere soil and other sources. In soil it 2007). Pseudomonas isolated from soyabean plant pro-
promotes direct plant growth and protects plants from the duced anti-fungal metabolites which inhibit the germina-
phytopathogens (Cook 1988; Glick 1995; Bashan and tion of mycelia of Sclerotina scletorum (Dilantha Fernando
Holguin 1998). The interaction between plants and bene- et al. 2005).
ficial Pseudomonas spp. exploiting bacterial traits for crop De Vleesschauwer et al. (2006) reported the use P.
protection has been well explained. The acquisition of aeruginosa 7NSK2 strain as a bio-control agent against the
information concerning the biosynthesis of the phenazine leaf blast (Magnaporthe grisea) and sheath blight (R. so-
molecules has almost exclusively been done through pyo- lani) in the monocot model rice plant. P. aeruginosa
cyanin produced by P. aeruginosa (Mercado-Blanco and 7NSK2 was treated in root which protected rice against leaf
Bakker 2007). blast leaving unprotected from sheath blight. The pyocya-
Ali Siddiqui et al. (2001) reported the use of rhizobac- nin produced by 7NSK2 enhanced the production of H2O2
teria in the control of root rot and root knot disease com- in roots and leaves, which degraded the toxic enzyme from
plex of mungbean. The organism isolated from the the plants. Onbasli and Aslim (2008) found that pyocyanin
rhizosphere was found to be P. aeruginosa which acted as a from Pseudomonas inhibits the E. coli isolates from sugar
bio-control agent in inhibiting the growth of Macropho- beet molasses.
mina phaseolina, Fusarium solani and Rhizoctonia solani. There are few cases in which the relative importance of
Pseudomonas also showed nematicidal activity in killing antibiotic production by bio-control bacteria like P. aeru-
the second stage larvae of Meloidogyne javanica. The ginosa has been demonstrated, where one or more genes
isolates from the soil were designated as P. aeruginosa IE- responsible for biosynthesis of the antibiotics have been
6 and P. aeruginosa Pa-7, -3, -4 and -33. Out of 33 isolates, manipulated. For example, mutant strains incapable of
17 of them exhibited antagonism against M. phaseolina, 14 producing phenazines (pyocyanin) or phloroglucinols have
were effective against F. solani and 18 inhibited the radial been shown to be equally capable of colonizing the rhi-
growth of R. solani. In greenhouse the effect of suppression zosphere but much less capable of suppressing soil borne
treatment of R. solani was observed, and the strain Pa-7 root diseases than the corresponding wild-type and com-
which was used in seed dressing inhibited R. solani to the plemented mutant strains. Bio-control strains are known to
maximum level. P. aeruginosa IE-6 was the most effective produce multiple antibiotics which can suppress one or
in reducing nematode penetration. P. aeruginosa Pa-7 more pathogens (Pal and Gardner 2006).
resulted in maximum plant height as well. Thus, P. aeru- Phenazines have been shown to influence the growth
ginosa not only acted as bio-control agents but also and to elicit induced systemic resistance (ISR) in plants
increased plant growth rate. (Pierson and Pierson 2010). Pseudomonas strains isolated
The role of pyocyanin from P. aeruginosa 7NSK2 from the rhizosphere plant were used to treat against var-
strain, in inducing resistance to Botrytis cinerea that causes ious species of Fusarium, Ralstonia and Meloidogyne
infection in tomato and grapevine was demonstrated by which cause wilting disease in coleus and ashwagandha
Audenaert et al. (2002). In their work, pyocyanin negative species (Mallesh 2008). The mechanism involved was
mutant strain and pyocyanin wild strain were used. In the studied and it was suggested that siderophore, HCN, indole
mutant strain PHZ l was coded by phzM gene. PHZ l was acetic acid, pyocyanin and other volatile metabolites syn-
unable to induce resistance and was co-inoculated with P. thesised by Pseudomonas strain exhibits the bio-control
aeruginosa 7NSK2 which restored resistance because of effect and they can also act as plant growth enhancers. Al
pyocyanin production by the strain. Anjaiah et al. (2003) Hinai et al. (2010) isolated umpteen number of P. aeru-
studied the use of pyocyanin from Pseudomonas species ginosa from greenhouse soils and all exhibited antagonistic
isolated from rhizosphere soil which controls Fusarium, effects against Pythium aphanidermatum which causes
the causative agent of wilt of chick pea and Pythium damping off of cucumber.
damping of bean. There have been studies on dual activity of pyocyanin
Reports on production of PCA produced by particular from P. aeruginosa TO3 as antibiotic against phytopatho-
strains of P. aeruginosa strain PUPa3 showed bio-control gen M. phaseolina and as a signalling molecule for
activity against a wide range of phytopathogenic fungi that development of biofilm by rhizobial strain Ca2. The
infect rice, groundnut, tobacco, chilli, mango, sugarcane, antagonistic activity of purified pyocyanin was determined

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by dry mass method which showed inhibition of M. capacity, including the ability to intrinsically resist antibi-
phaseolina and also biofilm formation by strain Ca2 was otics owing to its impermeable outer membrane, efflux
performed by crystal violet assay. There was an increase in capabilities, tendency to colonise surfaces in a biofilm form
biofilm development by Ca2 with an increase in pyocyanin and ability to acquire and maintain antibiotic plasmids
concentration up to 0.12 nmol/l. Using well-diffusion (Driscoll et al. 2007). Novel approaches for the treatment
method, the effect of pyocyanin on disease suppression and of P. aeruginosa infection are urgently needed.
biofilm formation by strain Ca2 on radicles of groundnut The ability of P. aeruginosa to cause disease is depen-
(Arachis hypogaea L.) was studied. A field study in soil dent upon the production of agents termed ‘virulence fac-
infested with M. phaseolina showed that a co-inoculant of tors’, such as toxins and adhesion molecules, that actively
P. aeruginosa TO3 and rhizobial strain Ca2 enhanced cause damage to host tissues. The concept of targeting
nodule mass and nitrogenase activity over that of the virulence using anti-infective or anti-virulence drugs that
control (Khare and Arora 2011). can ‘disarm’ the pathogenic bacteria rather than kill them
Despite their importance in biological control, little is has garnered increasing attention in recent years. It is
known about the genes involved in bio-control activity. believed that such an approach places less selective pres-
There has been a study in identifying the location of the sure on bacteria to evolve new strategies for survival,
antagonistic genes in P. aeruginosa FP6 towards phyto- thereby being more specific to pathogenic bacteria and
pathogens like R. solani and Colletotrichum gloeosporio- subject to less selection for drug resistance (Clatworthy
ides using PCR-based approach. A new bacterial strain, et al. 2007). In an analysis, it has been identified that
designated as FP6, was isolated from rhizospheric soil and pathogen-associated proteins have homologues only with
identified as a member of P. aeruginosa based on 16S pathogenic bacteria and not with non-pathogens (Ho Sui
rRNA analysis. The secondary metabolites produced by et al. 2009). Such proteins are more likely to have viru-
this strain have shown broad-spectrum bio-control activity lence-related functions. The list of pathogen-associated
against R. solani and C. gloeosporioides. The anti-metab- proteins identified included components of the phenazine
olites of a non-enzymatic nature were found to be biosynthesis pathway. Strains of P. aeruginosa produce
responsible for the antagonism. A chromosomal gene was and secrete a variety of redox-active phenazine com-
found to be involved in the production of antagonistic pounds, the most well studied being pyocyanin.
compound which was demonstrated by the gel elution Pyocyanin is considered both a virulence factor and a
technique using antibiotic gene-specific primers. The study quorum sensing (QS) signalling molecule for P. aerugin-
speculates that anti-metabolites play an important role in osa (Lau et al. 2004; Karatuna and Yagci 2010). QS-reg-
the control of phytopathogens (Bakthavatchalu et al. 2013). ulated virulence factors, and pyocyanin in particular, are
It was found that P. aeruginosa act as very good bio- active in lung infections associated with CF and produce
pesticides against soyabean stunt virus (SSV). P. aeru- numerous effects that are relevant to CF. Pyocyanin
ginosa has been isolated from rhizospheres of soyabean modulates redox cycling and generates reactive oxygen
and formulated in various forms such as liquid formulation, species capable of causing significant oxidative stress,
together with polyacrylamide hydrogel. The formulations which in turn affects calcium homeostasis. It inhibits cel-
were effective in plant growth and increased resistance lular respiration, depletes intracellular cAMP and ATP
against SSV also increases the yield, chlorophyll content levels, and thus affects chloride ion channels that are
and peroxidase activity. The study concluded that the controlled by ATP-driven conformational changes (Win-
application of P. aeruginosa formulation was effective stanley and Fothergill 2008). Pyocyanin inhibits prostacy-
against SSV (Khamdan and Suprapta 2011). clin release and can inactivate human V-ATPases
(involved in receptor-mediated endocytosis), a1-protease
inhibitor (which modulates serine protease activity,
Challenges and new insights into pyocyanin including neutrophil elastase) and nitric oxide (which
influences blood flow, blood pressure and immune func-
Pseudomonas aeruginosa is an opportunistic, Gram-nega- tions). Pyocyanin also alters the host immune response in
tive bacterium that causes infections in immune-compro- several ways to aid evasion of the immune system and
mised hosts, burn victims, individuals in intensive care and establish chronic infection. High concentrations of pyocy-
patients with CF. The lungs of nearly all CF patients are anin in the sputum of CF patients suggest that this com-
chronically colonised by P. aeruginosa, which significantly pound plays a key role in pulmonary tissue damage
reduces life expectancy and it is the leading cause of observed with chronic lung infections, and early (in the
morbidity and mortality for CF patients. The effectiveness growth curve) overexpression of QS regulated virulence
of P. aeruginosa as a pathogen can be attributed to its factors such as LasA, elastase and pyocyanin. These were
arsenal of virulence mechanisms and its large metabolic common among populations of a highly successful CF

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epidemic strain (Fothergill et al. 2007; Rada and Leto OdDHL-mimics with some previously-reported inhibitors
2013; Rada et al. 2013). Additional evidence demonstrates (based around different general structural frameworks) of
that pyocyanin significantly contributes to lung destruction QS from the literature, were also studied (Morkunas et al.
during chronic P. aeruginosa infection of bronchiectasis 2012).
airways in a mouse model and supports the hypothesis that There has been growing interest in disrupting bacterial
pyocyanin contributes to the accelerated decline in lung virulence mechanisms as a form of infectious disease
function of CF and other bronchiectasis patients once they control through the use of anti-infective drugs. There is a
are infected with P. aeruginosa (Caldwell et al. 2009). report on analysis of P. aeruginosa PAO1 which deduced
Pyocyanin biosynthesis is therefore an attractive target for proteome to identify pathogen-associated proteins. A
anti-infective drug intervention. computational screening approach was used to discover
Previously there has been investigative study on the drug repurposing opportunities, i.e. identifying approved
regulation of the phzA1B1C1D1E1F1G1 operon (phzA1). drugs that bind and potentially disrupt the pathogen-asso-
In an study conducted by Liang et al. (2011) screening of ciated protein targets. The selective oestrogen receptor
nearly 5,000 transposon mutants which revealed 14 inter- modulator raloxifene, a drug currently used in the pre-
rupted genes with altered phzA1 expression, including vention of osteoporosis and/or invasive breast cancer in
PA2593 (QteE), which has been identified as a novel reg- post-menopausal women, was predicted from this screen to
ulator of the QS system. Overexpression of qteE in P. bind P. aeruginosa PhzB2. PhzB2 is involved in produc-
aeruginosa significantly reduced the accumulation of tion of the pyocyanin. Raloxifene was found to strongly
homoserine lactone signals and affected the QS-controlled attenuate P. aeruginosa virulence in a Caenorhabditis
phenotypes such as the production of pyocyanin, rhamn- elegans model of infection. Treatment of P. aeruginosa
olipids, LasA protease and swarming motility. Indeed, wild-type strains PAO1 and PA14 with raloxifene resulted
overexpression of qteE in P. aeruginosa attenuated its in a dose-dependent reduction in pyocyanin production
pathogenicity in the potato and fruit fly infection models. in vitro; pyocyanin production and virulence were also
These findings suggest that qteE plays an important role in reduced for a phzB2 insertion mutant. The results suggest
P. aeruginosa pathogenicity and is part of the regulatory that raloxifene may be suitable anti-infective or anti-viru-
networks controlling phenazine production. lence agents for further development as a therapeutic for P.
Hassani et al. (2012) reported nearly eighty-two mutants aeruginosa infection (Ho Sui et al. 2012).
isolated from wild type strain of P. aeruginosa PHA-1 Bacterial adhesion and biofilm formation are both
using various concentrations of cefotaxime. Wild type dependent on the production of extracellular polymeric
PHA-1 and mutants were examined for production of py- substances (EPS) mainly composed of polysaccharides,
ocyanin. Remarkably, the mutant strain named S300-8 was proteins, lipids, and extracellular DNA (eDNA). eDNA
distinguished in productivity in comparison with wild type promotes biofilm establishment in a wide range of bacterial
strain PHA-1. In addition, pyocyanin produced by mutant species. In P. aeruginosa, eDNA is major component of
strain S300-8 revealed a potent efficacy against growth of biofilms and is essential for the formation and stability. Das
cancer cell line RD; the low concentration of this pigment and Manefield (2012) in their study reported that produc-
caused 65 % of dead cells after 72 h of incubation whereas tion of pyocyanin in P. aeruginosa PAO1 and PA14 batch
the cytotoxicity was improved by increasing the concen- cultures is responsible for promotion of eDNA release. A
tration of pigment with period of exposure time. phzSH mutant of P. aeruginosa PAO1 that overproduces
Pseudomonas aeruginosa regulates pyocyanin produc- pyocyanin displayed enhanced hydrogen peroxide (H2O2)
tion using an intercellular communication mechanism generation, cell lysis, and eDNA release in comparison to
mediated by small signalling molecules termed as auto- its wildtype strain. A DphzA-G mutant of P. aeruginosa
inducers. One native auto-inducer is N-(3-oxododecanoyl)- PA14 deficient in pyocyanin production generated negli-
L-homoserine lactone (OdDHL). There are reports about gible amounts of H2O2 and released less eDNA in com-
the synthesis of collection of abiotic OdDHL-mimics. A parison to its wildtype counterpart. Pyocyanin intercalation
number of novel compounds capable of competing with the with eDNA promotes cell-to-cell interactions in P. aeru-
endogenous OdDHL and consequently, inhibiting the pro- ginosa cells by influencing their cell surface properties and
duction of pyocyanin in cultures of wild type P. aeruginosa physico-chemical interactions (Das et al. 2013).
were identified. The evidence suggest that the compounds Using the combination of microarray and metabolite
of this general structural type act as direct antagonists of analyses, Lundgren et al. (2013) demonstrated about the
QS in P. aeruginosa and as such may find value as assimilation of glycine as a carbon source and the bio-
molecular tools for the study and manipulation of this synthesis of pyocyanin in P. aeruginosa PAO1; both are
signalling pathway. Direct quantitative comparison of the dependent on the PA2449 gene. The inactivation of the
pyocyanin suppressive activities of the most active PA2449 gene was found to influence the set of core genes

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of transcription-encoding glycine cleavage system, serine chemical pesticides. Further new insights about pyocyanin
hydroxymethyltransferase, and serine dehydratase. PA2449 are building up in the field for new research opportunities.
also affected the transcription of several genes that are
integral in cell signaling and pyocyanin biosynthesis in P. Acknowledgments The authors are thankful to all the researchers
whose papers have been used for this review. Greatest gratitude goes
aeruginosa PAO1. to Luigi Lucini, Ph.D., Università Cattolica del Sacro Cuore, Institute
Natural products have recently become a promising of Environmental and Agricultural Chemistry.
source for deriving molecules that can potentially inhibit
QS related anti-virulent activities. Reports about screening
of the QS inhibitory properties of 16 high quality ayurvedic
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