The Role of Phenazines as an Electron Transporter in Pseudomonas aeruginosa Biofilm and its Effect on Colony Morphology

Huy PhamMicro 496 Paper

Introduction:

One of the major challenges facing medical care of modern times is chronic infection of

immunocompromised patients by opportunistic pathogens, like pseudomonas. The gram-negative

Pseudomonas aeruginosa is thought to be strictly aerobic, and is known to infect the respiratory

tract of cystic fibrosis patients and persist via development of biofilm. Once established, the

biofilm plays an important role in sheltering the bacterial colony from environmental factors like

desiccation and the host’s immune system. It is noted that oxygen availability at the center of the

biomass (biofilm) is lower than the expected threshold for which strict aerobes can grow (3),

which leads to the question: what factor(s) can account for the thriving of Pseudomonas in

microaerobic conditions?

Biofilm development illustration. D. Davis, 2007

It is a well known phenomena, frequently observed in cystic fibrosis patients, that

pseudomonas in critical mass, form biofilms via quorum-sensing cascades (5), and secrete

phenazines into the extracellular environment. A readily observed property of phenazine is its

redox-active toxicity to non-Pseudomonas organisms (4), and can be considered an antibiotic.

Phenazine can also be observed as an active electron transport shuttle in the biomass as well,

which can promote O2-independent survival in microaerobic environments, such as the depths of

the biomass. One area of research is looking at the potential link between pyocyanin, a class of

phenazine, and its role in facilitating pseudomonas respiration in the microaerobic region of

biofilms. In recent studies, it has been shown that mutant Pseudomonas with knockout phenazine

expression (∆-phenazine) generate less robust colonies than its wild-type variant. This result

indicates that phenazine plays a crucial role in the survival and growth of Pseudomonas biomass

via extracellular electron transfer, and contributes to their virulence. This paper will look at

studies that examine the possible association between pyocyanin as an electron transport shuttle,

and the ability of Pseudomonas to persist and grow in a microaerobic environment.

The role of phenazine in bacteria, and pyocyanin in pseudomonas:

As noted in the review article, “Metabolism and

function of phenazines in bacteria: impacts on the

behavior of bacteria in the environment and biotechnological process,” (5) phenazines are a

group of nitrogen-containing heterocyclic compounds that differ in function and properties based

on type and position of functional groups present. Hundreds of phenazine derivatives have been

identified in nature. Bacteria are the only known producer of phenazine in the natural world.

Derived from the shikimic acid pathway, a highly conserved metabolic feature in most

organisms (5), recent studies on phenazines have shown their role in inducing plant defense

pathways, electron transportation, iron chelation, biofilm formation, and even gene expression.

Phenazines are known to form reactive oxygen species, and have been implicated in bacterial

virulence, and can confer competitive advantages over competing organisms as an

antifungal/antibiotic.

The phenazine of interest, pyocyanin, first isolated from patient wounds (5), is

synthesized by the bacterium Pseudomonas aeruginosa, and regulated by quorum sensing

signals. Pyocyanin have been implicated in the inhibition of competing microorganisms during

mixed species lung infections. In addition to acting as a competitive antibiotic, pyocyanin is

found to be secreted into the extracellular region of the pseudomonas biofilm, and research have

assayed its role as an electron transport shuttle, as discussed in the next section.

Studies on pyocyanin as an extracellular electron transport shuttle in Pseudomonas aeruginosa:

One of the experimental studies that showed how Pseudomonas is able to maintain redox

homeostasis under O2-limited conditions was published in 2007, called, “Pyocyanin Alters

Redox Homeostasis and Carbon Flux through Central Metabolic Pathways in Pseudomonas

aeruginosa.” (6)

In the first part of the study, researchers assayed the oxidized versus reduced state of

pyocyanin as catalyzed by stationary phase LB cultures of Pseudomonas. When left undisturbed

for five minutes, the pyocyanin-laced suspension turned clear, which is indicative of pyocyanin’s

reduced state. When vortexed, the same pyocyanin suspension turned deep blue, which is

indicative of its oxidized state.

Left to Right:Tube 1: Exponential-phase LB culture.Tube 2: Stationary-phase LB culture, immediately after removal from shaking incubator.Tube 3: Stationary-phase LB culture, left standing at room temperature for 5 minutes.Tube 4: 100µM pyocyanin in MOPS buffer.Tube 5: Same culture as in tubes 2 and 3, resuspended in buffer show in tube 4 and left standing at room temperatureTube 6: Same suspension as in tube 5, after vortexing

It is noted that at the air-liquid interface, the suspension is always blue, a good

implication of pyocyanin oxidation by O2. This experimental data suggest that pyocyanin in its

clear, reduced state, is oxidized by an abiotic reaction with oxygen, as a result turning blue, and

when left undisturbed for five minutes, is reduced once again to a clear color by the act of

Pseudomonas donating electrons, as demonstrated by Tube 5. This cycle of oxidation and

reduction can be repeated as many times as desired via resting followed by vortexing, as

demonstrated by Tube 6, to show that phenazine is able to continuously cycle between oxidized

and reduced form, thus making it a suitable electron-transporting candidate.

With prior knowledge that NADH reacts with pyocyanin in vitro, researchers

hypothesized that the presence of pyocyanin would decrease intracellular NADH levels. In the

second part of the study, researchers attempt to measure intracellular NADH/NAD+ ratio in wild-

type versus ∆-phenazine mutant, both of which were in stationary growth phase. The

NADH/NAD+ ratio in the wild-type were less than half of that observed in the ∆-phenazine

mutant. After this observation, pyocyanin was added to the ∆-phenazine mutants, resulting in a

decrease of NADH/NAD+ to wild-type level. Knowing that phenazine can accept electrons from

NADH, it can be inferred from these observations that the intracellular NADH/NAD+ ratio is

influenced by the availability of redox reagents.

To further confirm the findings above, the third part of the study involved assaying

NADH/NAD+ ratio and pyocyanin production in an increasingly O2-depleted environment. The

researchers grew batch cultures in a fermentor at 37C, which allowed them to control

temperature and aeration of the suspension. As O2 levels plummeted in the wild-type batch,

pyocyanin production sharply increased, and a decrease in NADH/NAD+ followed. The previous

decrease in NADH was not observed in the ∆-phenazine mutant; as O2 plummeted, no pyocyanin

was produced, and NADH/NAD+ remained high. Without phenazine in a hypoxic environment,

it became clear that the cells will not have an electron shuttle for redox activities. The outcomes

of this particular experiment demonstrated the importance of pyocyanin in extracellular redox

activities in hypoxic conditions.

Studies on the pyocyanin effect on biofilm morphology:

The previous study has shown evidence that Pseudomonas is capable of extracellular

redox activity through the usage of pyocyanin as an electron shuttle, but what outcome will this

phenomena have on the properties of biofilm, and how does it affect the virulence of the

pseudomonas colony?

An experimental study published in 2008, “Redox-active antibiotics control gene

expression and community behavior in divergent bacteria,” (2) assayed the morphological aspect

of colony development of various strains of pseudomonas with varying levels of pyocyanin

production. The strains involved in the experiment were: wild-type Pseudomonas aeruginosa

strain 14; ∆-phenazine, which has knockout phenazine-encoding genes; Dianne K. Newman

strain 370 (DKN370), which overproduces phenazine; and two other pseudomonas mutant

strains as explained below.

Pyocyanin, in addition to being a redox reagent, is also responsible for the up-regulation

of over 35 genes (2), including the SoxR and mex operon in pseudomonas, which produces

putative monooxygenase upon activation, and efflux pumps respectively. Given that fact,

pyocyanin’s dual role as an extracellular redox reagent and gene expression modulator will

complicate the result in answering the question of how pyocyanin, specifically as an electron

transport shuttle, will directly affect the morphology of the colony. To partially address this

complication, the researchers have included a strain of pseudomonas with SoxR-knockout (∆-

SoxR) and mex-knockout (∆-mex) mutation to partially eliminate gene modulation, and isolate

the electron transport aspect of pyocyanin in the experiment.

35 colonies of each strain was grown on agar plates at room temperature and observed

over eight days. The colony traits observed included surface area coverage, and

wrinkliness/smoothness and pigmentation. Surface area coverage and wrinkliness is an

adaptation to hypoxia (3), and implicative in the reaction of a bacterial colony to oxygen

demand. It is known that oxygen is used up quickly in the colony, and even at shallow depths,

the biofilm environment approaches hypoxia. Given the high organism density and oxygen

demand in the biofilm, the colony arranges itself via quorum sensing factors to maximize oxygen

intake by maximizing surface area exposure. The effect the pigmentation has on the colony

morphology in this experiment is not well understood, and further development is needed to

better understand the mechanisms involved in this expression.

The experimental results, as seen above, shows increasing surface area and wrinkliness of colony

morphology as time elapses, though with varying degrees based on strains. The wild-type colony

shows a steady progression towards increasing wrinkliness as cells rearrange themselves for

maximum oxygen exposure. ∆-phenazine exhibit the most advanced wrinkliness and surface area

coverage as it spreads out and tries to compensate for lack of extracellular electron shuttle. Note

that it is evolutionarily undesirable for colonies to exhibit wrinkliness, as it exacerbates

desiccation (3). DKN370, with its overproduction of pyocyanin, stayed the smoothest. ∆-SoxR

and ∆-mex was measured to have released more pyocyanin into the agar than wild-type, making

them behave similarly to the pyocyanin overproducer DKN370. In this experiment, there appears

to be a direct correlation between the quantity of extracellular pyocyanin and colony smoothness,

which is inferred to be evolutionarily advantageous in terms of desiccation resistance and

survival in hypoxic environment.

The mechanism dictating the phenotypic effects of pigmentation on the colony

morphology is not well understood (2), but because the ∆-phenazine colony is bright red, and the

pyocyanin overproducer DKN370 is pale, it could be inferred that there is an inverse relationship

between pigment production and pyocyanin secretion. There is much to be researched on such a

relationship before a plausible link can be established.

What can be done to further develop hypothesis:

Given our biochemical understanding of how redox reaction works with phenazine, and

the biological plausibility of pyocyanin’s role as an extracellular electron transporter. It can be

strongly inferred that pyocyanin plays a key role as redox reagent in hypoxic environment, as

demonstrated by the redox assay in the first experiment (6). The second study of the effects of

pyocyanin on colony morphology also supported the extracellular redox reagent hypothesis, but

it also opened up a few unanswered questions.

The second study (2) showed that pseudomonas form more robust biofilms with

heightened expression of extracellular phenazines. Knowing that phenazine has more than one

role in the virulence of pseudomonas, the experiment raises more questions about the actual

mechanism involved in the maintaining robustness of the biofilm: how much of the robustness of

the biofilm is attributable to pyocyanin as an electron transporter, and how much of it due to the

modulation of gene expression as a result of its role as a quorum sensing signal?

Of pyocyanin’s regulation of gene expression in pseudomonas, many up-regulation

pathways are currently not well-understood, such as pigmentation production, and therefore not

accounted for, and further advancement is needed to progressively account for the subtle process

in which these genes promote virulence in the organism.

One proposal to indirectly understand the role of pigmentation in the context of gene

regulation is to measure its production in response to various stimuli. An untested hypothesis that

I propose, is that the pigmentation also serve as a response to oxidative stress. There is a

relatively strong association between wrinkliness and pigmentation production that I have

observed from the colony morphology experiment. Perhaps one possible experiment to

determine pigmentation production in response to oxidative stress is to expose strains of

pseudomonas, and their mutant variants, to increasing oxygen saturation, and observe the colony

pigmentation and morphology as time elapses. This experiment may indirectly account for the

role pigmentation play in pseudomonas, and as a result, better resolve the relationship between

exposure to phenazine and colony morphology by ways of better accounting for pigmentation.

Once the up-regulations mechanisms are better mapped out, the experiment may be

improved by introducing new knockout mutations that will more directly assay pyocyanin’s

properties while accounting for its many subtle regulations of gene expression.

Conclusion:

Studies on phenazine as an electron transport and its effect on colony morphology

indicate that there is a causal link between the former and latter. Phenazines are needed as

electron acceptors in hypoxic environments. Pseudomonas colonies that lack pyocyanin cannot

survive well in hypoxic environments, thus forming not-so-robust biofilms that are subject to

desiccation. Multiple studies demonstrate numerous metabolic pathways that involve pyocyanin

beyond the focus of this paper (5), implicating that they serve even more important functions in

the competitive survival of their host than just extracellular electron transport. All of these

studies point to a strong association between phenazines and the evolutionary advantage it

confers to biofilm and the virulence of bacterial colonies.

Now that there is strong evidence showing that phenazine promotes virulence in

pseudomonas by ways of redox, the researchers now need to account for the various regulations

of genes under the promotion of pyocyanin. The colony morphology experiment showed not

only results that further support the pseudomonas extracellular electron transport hypothesis, it

also showed pigmentation changes as a secondary effect of pyocyanin production that is yet to be

accounted for. The difficulty in showing the mechanism of the regulation pathways is that we

still do not know much about how the cohorts of operons interact with one another; it is still

work in progress and there is much research to be done in this field.

The ultimate goal of studies in this field is to progress new findings that will better

resolve current questions about pseudomonas virulence, ask better questions about unresolved

operons, and ultimately lead to better antibiotic lines and treatment of immunocompromised

patients. Pyocyanin, as shown by pseudomonas, can be used not unlike penicillin, to inhibit other

organisms from growing (4), and its derivatives holds good potential in medical and industrial

applications.

Bibliography

1. D. Davis. “5 Stages of Biofilm Development.” March 2007 Doi:info:doi/10.1371/journal.pbio.0050307.g001

2. Dierich, Lars E. P. et al. “Redox-Active Antibiotics Control Gene Expression and Community Behavior in Divergent Bacteria.” Science, 29 August 2009; 321(5893); 1203-1206.

3. Newman, D. K. “From Iron Oxides to Infections: Roles for Redox-Active Antibiotics in Microbial Survival and Development.” T-639 Auditorium, UW-Seattle. 27 Feb 2012. Lecture

4. Park, GK et al. “Elucidation of antifungal metabolites produced by Pseudomonas aurantiaca IB5-10 with broad-spectrum antifungal activity.” J Microbiology Biotechnology, 2012 Mar; 22(3): 326-30

5. Pierson, Leland S. III et al. “Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes.” Published online at Springerlink.com, 30 March 2010.

6. Price-Whelan, Price et al. “Pyocyanin Alters Redox Homeostasis and Carbon Flux through Central Metabolic Pathways in Pseudomonas aeruginosa PA14.” Journal of Bacteriology, 24 May 2007; 189(17):6372.

7. Ramos, Itzel et al. “Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales.” Res. Microbiol., 16 April 2010; 161(3): 187-191.

8. Wang, Yun et al. “Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer.” Journal of Bacteriology, 30 October 2009; 192(1):365.