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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.
References• Chaudhari V, Gosai H, Raval S and Kothari V. (2014). Effect of certain natural products and organic
solvents on quorum sensing in Chromobacterium violaceum. Asian Pac J Trop Med, Suppl1,S201-S211.• Darshan N and Manonmani HK. (2015) .Prodigiosin and its potential applications. Journal of Food
Science and Technology. Vol. 52, p5397-5407.• Elahian F, Moghimi B, Dinmohammadi F, Ghamghami M, Hamidi M and Mirzaei SA. (2013). The
anticancer agent prodigiosin is not a multidrug resistance protein substrate. DNA and Cell Biology. Vol32, p90-97.
• Lapenda JC, Silva PA, Vicalvi MC , Sena K X and Nascimento S C. (2015). Antimicrobial activity of prodigiosin isolated from Serratia marcescens UFPEDA 398. World journal of Microbiology and Biotechnology. Vol31,p399–406.
• Mekhael R and Yousif S Y. (2009) . The role of red pigment produced by Serratia marcescens as antibacterial and plasmid curing agent. J.Duhok Univ. Vol12,p268-274.
• 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.
• Montaner B, Navarro S, Piqué M, Vilaseca M, Martinell M, Giralt E, Gil J, Pérez-Tomás R (2000). Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Vol.131, 585-593.
• Rahul S, Chandrashekhar, Hemant B, Bipinchandra S, Mouray E, Grellier P and Satish P. (2015). In vitro antiparasitic activity of microbial pigments and their combination with phytosynthesized metal nanoparticles .Parasitology International.
• Song MJ, Bae J, Lee DS, Kim CH, Kim JS, Kim SW and Hong SI. (2006). Purification and characterization of prodigiosin produced by integrated bioreactor from Serratia sp. KH-95. Journal of Bioscience and Bioengineering, Vol. 101, p157–161.