Isolation and Characterization of Prodigiosin from Soil Bacterium

Under the guidance of-• Dr. K. Kumanan (External guide; Dean, Faculty of Basic Sciences, TANUVAS, Madras Veterinary College, Chennai.)• Mrs. G. Dhanavathy (Internal guide; Assistant Professor, Department of Biotechnology, SRM University, Chennai.)

Efforts by-• Aditya Sharma (1091210015)• Aditya Rishi (1091210038)• Ashley Abraham (1091210040)

Introduction

•Serratia marcescens is a gram-negative, rod-shaped, motile bacterium belonging to the family Enterobacteriaceae. •S.marcesescens are facultative anaerobes that produce a red to dark pink pigment called prodigiosin which is an alkaloid secondary metabolite with a unique tripyrrole chemical structure. •It has been reported to exhibit anti-bacterial, anti-fungal, anti-neoplastic, anti-proliferative, anti-oxidant , immunosuppressive and anti-malarial activities.•DNA staining ability of prodigiosin has the potential to substitute the chemically synthesized , carcinogenic, conventional stains like ethidium bromide.

Plate.1: Prodigiosin((5( (3-methoxy- 5-pyrrol-2-ylidene-pyrrol-2-ylidene) -methyl) -2- methyl-3-pentyl-1H- pyrrole)

Nat. Rev. Microbiol., Williamson et al, 2006

Review of Literature• Serratia marcescens is known to produce more concentration of prodigiosin when compared to other

strains like Pseudomonas , Streptomyces, Pseudoalteromonas sp., Haella chejuensis and Vibrio sp.(Song et al., 2006; Blake et al. 1990)

• The regular liquid media currently being used for prodigiosin biosynthesis is nutrient broth .(Pryce and Terry, 2000)

• The optimum production of prodigiosin was at 28◦C after 48 hrs. (Siva et al.,2012; Darshan and Manomani, 2015)

• Colour change of prodigiosin extract to red in acidified sol. and yellow/tan in alkaline sol. gives a positive presumptive test for prodigiosin (Gerber and Lechevalier, 1976).

• Thin layer chromatography of prodigiosin was carried in hexane-methanol mobile phase, Rf value deduced to be 0.8 (Rahul et al., 2016).

• HPLC of prodigiosin was carried in orthophosphoric acid-acetonitrile mobile phase, retention time observed was 10.424 min (Chaudhari et al., 2014).

• The Serratia marcescens prodigiosin showed bactericidal and bacteriostatic effect showing promising antimicrobial activity.(Lapenda et al.,2014)

• Prodiginines have been described as pro-apoptotic anticancer compounds and have been shown to induce cellular stresses such as cell cycle arrest, DNA damage and a change of intracellular pH (pHi), all of which can induce apoptosis (Darshan and Manonmani, 2015).

• Prodigiosin is shown to intercalate into dsDNA. In the presence of copper, it promotes oxidative cleavage of the DNA. (Melvin et al., 2002)

• In in-vitro and cultured cells, prodigiosin-DNA interaction is shown to prefer alternating base pairs but with no discrimination between AT and GC sequences; dual abolition of topoisomerase I and II activity and as a consequence, DNA cleavage. (Montaner et al., 2005)

Objectives

• To isolate the pigment prodigiosin from soil bacterium Serratia marcescens.• To characterize prodigiosin.• To test prodigiosin for its anti-microbial activity.• To test prodigiosin for its anti-neoplastic activity.• To test prodigiosin for its DNA staining ability.

Methodology

Pigment production and extraction

Estimation of prodigiosin by UV spectrophotometry

Characterization of pigment using TLC and HPLC

MTT assay performed on A-72, Hep-2 and Vero cell lines

Biochemical analysis (presumptive test) for preliminary confirmation

Determination of DNA staining ability by agarose gel electrophoresis

In-vitro determination of anti-microbial activity using well diffusion method

Results

Plate.2: Serratia marcescens culture in nutrient broth

Plate.3: Serratia marcescens streaked nutrient agar plates showing discrete colonies

Plate.4: Prodigiosin pigment isolated from S.marcescens in 4% acidified ethanol

Plate.5: 2 ml of lyophilized prodigiosin pigment isolated in 4% acidified ethanol

Plate.6: Colour change of prodigiosin extract to red in acidified sol. Aand yellow in alkaline sol., showing a positive presumptive test for prodigiosin.

T1: Prodigiosin extract acidified with a drop of conc. HCl.T2: Prodigiosin pigment isolated from S. marcescens in 95% methanol (extract).T3: Prodigiosin extract alkalinized with a drop of conc. ammonia sol.

Results

Prodigiosin unit/cell = [OD499 – (1.381 x OD620)] x 1000 OD 620

Serratia marcescens was found to produce 1516.9 prodigiosin units/cell in nutrient broth.

Estimation of prodigiosin by UV spectrophotometer:Isolated prodigiosin was estimated using the following equation (Mekhael and Yousif, 2008) : OD499 – pigment absorbance

OD620 – bacterial cell absorbance 1.381 – constant

Prodigiosin unit/cell =

[0.823 – (1.381 x 0.284)] x 1000 0.284

= 1516.9 units/cell

T1 T2 T3

OD499 (pigment absorbance) OD620 (bacterial cell absorbance)

0.807 0.2740.820 0.2880.842 0.291Average = 0.823 Average = 0.284

Fig.1: HPLC analysis of prodigiosin showing a retention time of 10.757 min. (closely corresponding to the retention time of 10.424 min.(Chaudhari et al., 2014))

ResultsPlate.7: Thin layer chromatography of pigment prodigiosin in hexane:methanol (1:2) mobile phase , exhibiting an Rf of 0.81 (In agreement with Rf of 0.8 deduced by Rahul et al. (2015) in the exact solvent system)

Plate.8: Assay showing the zone of inhibition at 25 µg, 50 µg and 75 µg concentration of the pigment prodigiosin against few bacterial strains

Streptococcus aureus Salmonella enterica Escherichia coli Staphylococcus agalactiae

Results

 

Strep

tococ

cus a

ureu

s

Staph

yloco

ccus

agala

ctiae

Salm

onell

a enter

ica

Eschire

chia

coli

05

10152025

Antimicrobial activity of varying prodigiosin concentration – A comparison

Control

25 µg

50 µg

75 µg

Bacterial species

Zone

of i

nhib

ition

in m

m

Fig.2: Antimicrobial activity of varying prodigiosin concentration - A comparison.

Fig.3: MTT assay of A-72cells indicating cytotoxicity of prodigiosin. Increased cell death was noticed when the concentration of prodigiosin was increased

Fig.4: MTT assay of Hep-2 cells indicating cytotoxicity of prodigiosin. Increased cell death was noticed when the concentration of prodigiosin was increased

Results

Fig.5: MTT assay of Vero cells indicating cytotoxicity of prodigiosin. Increased cell death was noticed when the concentration of prodigiosin was increased

Fig.6: A comparison showing specific neoplasm directed cytotoxicity of prodigiosin

Cancer cells contain 3.5 fold higher concentration of Cu(II) than non-malignant cells

The electron rich bipyrrole chromophore of permeable prodigiosin oxidizes itself

In turn reduces stable Cu(II) to unstable Cu(I)

Cu(I) then inhibits phosphorylation of enzymes- topoisomerase I & II , hence inactivating the enzymes

This causes super-coiling of DNA leading to its fragmentation

Results

5 µg EtBr

2 µg prodigiosin 3 µg prodigiosin 5 µg prodigiosin 10µg prodigiosin4µg prodigiosin

20 µg prodigiosin 30 µg prodigiosin 60 µg prodigiosin50 µg prodigiosin40µg prodigiosin

L1: 1 kb DNA ladderL2: Staphylococcus agalactiae DNAL3: Escherichia coli DNAL4*: Love bird DNA

Plate.9: Electrophoresis of DNA samples in 0.8% agarose gel stained with increasing concentration of prodigiosin

Summary• Nutrient broth was found to be the optimum medium to produce prodigiosin. (1516.9

prodigiosin units/cell in nutrient broth , greater than that produced in triolein , maltose containing medium and dextrose).

• The natural red pigment produced by S. marcescens demonstrated a retention factor and retention time corresponding to prodigiosin reported in the literature in addition to

a positive presumptive test.

• The clinical isolates evaluated in our study- Streptococcus aureus, Staphylococcus agalactiae, Salmonella enterica and Eschirechia coli, exhibited relevant sensitivity to prodigiosin as significant inhibition zones were achieved.

• A dose-dependent decrease in the number of viable cells was observed in all the cell lines studied . However, we did not observe a significant decrease in the viability of the non-malignant Vero cells when compared with the malignant cell lines. Hence, it can be inferred that prodigiosin possesses specific neoplasm directed cytotoxicity .

• It was observed that prodigiosin starts staining DNA at a concentration as low as 3 µg. However, effective staining was observed at a range between 10 µg and 40 µg. At 50 µg concentration, agarose gel electrophoresis showed DNA fragmentation, due to the positive correlation between cytotoxicity and DNA cleavage. 

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• Melvin M, Tomlinson J, Saluta G, Kucera G, Lindquist N and Manderville R. (2000). Double-strand DNA cleavage by copper prodigiosin. Journal of American Chemical Society. Vol.122,p6333-6334.

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