Transcript bsk viva ppt - bsk hates diversity & differences..

IN SILICO COMPARISON OF XENOBIOTIC
DEGRADATION PATHWAYS AMONG THREE
STRAINS OF PURPLE NON SULFUR
BACTERIA AND CONFIRMATION OF
ANILINE DEGRADATION BY
RHODOBACTER SPHAEROIDES DSM 158
BY
KARTHIKEYAN.B.S
BI0807
UNDER THE GUIDANCE OF
DR.CH. SASIKALA,
Associate Professor, Bacterial Discovery Laboratory,
Centre For Environment, Institute of Science and Technology,
Jawaharlal Nehru Technological University, Hyderabad
PROJECT SUPERVISORS
Dr. Ch .Sasikala M.Sc.,Ph.D
Associate Professor,
Bacterial Discovery Lab,
Center for Environment,
Institute of Science and Technology,
Jawaharlal Nehru Technological
University,
Hyderabad.
Dr.Ch.Venkata Ramana, M.Sc.,Ph.D
Professor,
Anoxygenic Phototrophic Bacterial Lab,
Department of Plant science,
School of Life Science,
University of Hyderabad,
Hyderabad.
BIOINFORMATICS
COMPARATIVE
GENOMICS
METABOLIC
DATABASES
BACTERIAL
DISCOVERY
LABORATORY
MICROBIOLOGY
METABOLOMICS
XENOBIOTIC DEGRADATION
OBJECTIVES OF THE STUDY
•
•
•
Rhodopseudomans palustris CGA 009
Rhodobacter sphaeroides 2.4.1
Rhodospirillum rubrum ATCC 17001
In Silico:
1. To compare ability of three strains to degrade xenobiotic compounds
2. To select the efficient strain out of three strains for efficient Xenobiotic
degradation
3. To select the substrate or xenobiotic compound for a single selected strain to
carry out in vivo experiments
In Vivo:
4. To study the effect of the selected substrate on the growth of the selected
strain
5. To determine the capability of the selected strain to degrade the selected
substrate
6. To determine the capability of the selected strain to transform the selected
substrate into different metabolites
HIERARCHY OF WORK CARRIED OUT
WHOLE GENOMES OF APB (Rhodopseudonomanas palustris
CGA009,
Rhodobacter sphaeroides DSM 158, Rhodospirillum rubrum ATCC
11170)
GENOME WIDE
COMPARISONS
(IMG JGI)
% OF XENOBIOTIC DEGRADATION
CODING GENES IN ALL THE
SELECTED STRAINS
SEQUENCE BASED
COMPARISONS
(KEGG, METACYC)
COMPARISON BETWEEN
SPECIES
% OF ANNOTATED GENES CODING
FOR XENOBIOTIC DEGRADATION
IN SILICO
RESULT
IN SILICO
RESULT
Cont..
SELECTION OF STRAIN AND
SUBSTRATE
ACCORDING TO AVAILABILITY OF
STRAIN AND CHEMICAL
REVIVING OF THE CULTURE & CHECKING
THE PURITY
GROWTH WITH CHEMICAL AS C OR
N SOURCE (OD 660)
GROWTH YIELD (OD 660)
DISAPPEARANCE OF THE CHEMICAL
(UV SPECTROPHOTOMETER)
ANALYSIS OF THE RESULTS
DEGRADATION OF CHEMICAL AT
OPTIMUM CONCENTRATION USING
HPLC
IN VIVO
OPTIMUM CONCENTRATION
SELECTED
QUALITATIVE ANALYSIS OF PRODUCTS
OF DEGRADATION THROUGH HPLC
ESTIMATION OF INDOLE
ANOXYGENIC PHOTOTROPHIC BACTERIA
•
XENOBIOTIC COMPOUNDS & ITS PROPERTY
- man made chemicals,, hazardous to living beings.
•
BIODEGRADATION & ITS ROLE
- effective, minimally hazardous, and economical.
•
MICROBES AND ITS EFFICIENCY
- microorganisms exist billions of years
- survived with variety of organic compounds for energy etc.,
•
BIOINFORMATICS FOR BIOREMEDIATION
- microbial degradation pathways is so incomplete
- explore new catabolic pathways
•
GENOMICS FOR BIOREMEDIATION
- microbial genes to evolve mechanisms to degrade synthetic organic
structures
Cont..
- potential metabolic activity of the microbial community
- isolated organisms were important in bioremediation or not ?
• NEED FOR SYSTEMS APPROACH
- complex interactions between cellular reactions from a genomic and
proteomic level
- system biology approach is necessary to predict the functioning of an
organism in a complex environment and to describe the outcome of
the thousands of individual reactions that are simultaneously taking
place in a microbial cell.
• ADVANTAGES OF USING BACTERIA
- many completed whole genomes are available
- most numerous and obvious microbial components of the earth
- easy to culture
- ease to integrate in silico and in vivo approaches
Cont..
•
ANOXYGENIC PHOTOTROPHIC BACTERIA (APB)
- photosynthetic prokaryotes
-anaerobic conditions by photosynthesis with out oxygen liberation
- lack photo system-II and carryout anoxygenic photosynthesis
•
Purple Non-Sulfur Bacteria
- metabolize wide range of aliphatic organic compounds
- used in sewage treatment
•
Rhodopseudomonas palustris
- extraordinary metabolic versatile and successful metabolic opportunist
- photoautotrophic, photo heterotrophic, chemo heterotrophic , chemoautotrophic
metabolism
- encodes four distinct oxygenase-dependent ring cleavage pathways
- well studied for aromatic compound degradation.
•
Rhodobacter sphaeroides
- metal reduction, nitrogen fixation, hydrogen production
- microaerophilic conditions, chemotropic and phototrophic growth
•
Rhodospirillum rubrum
- production of biological plastic (PHB poly-hydroxy-butric-acid) , nitrogen fixation,
biofuel production.
MATERIALS AND METHODS IN SILICO
•
GENOME INFORMATION
- Integrated Microbial Genomes (IMG) system and KEGG database
•
WHOLE GENOME SEQUENCES
•
WHOLE GENOME COMPARISONS
- xenobiotic degrading genes present in the genome
- comparison between the other strains
•
GLOBAL MAP OF ENTIRE METABOLISM
- KEGG pathway database
•
XENOBIOTIC PATHWAYS
- KEEG
- METACYC
•
SEQUENCE COMPARISON OF XENOBIOTIC DEGRADING GENES
•
SELECTION OF THE STRAIN FOR DEEPER ANALYSIS
•
PUTATIVE ENZYMES OF RHODOBACTER SPHAEROIDES DSM 158
•
SELECTION OF SUBSTRATE AND PATHWAY FOR DEGRADATION
•
MINING OF NAPHTHALENE AND ANTHRACENE DEGRADATION
MATERIALS AND METHODS IN VIVO
• PURIFICATION
• PREPARATION OF MEDIA
Cont..
•
DETERMINATION OF GROWTH AND WHOLE CELL ABSORPTION SPECTRUM
•
EFFECT OF ANILINE ON NORMAL GROWTH
•
GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF
ANILINE AS CARBON AND NITROGEN SOURCE (ANAEROBIC)
•
GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN PRESENCE OF
ANILINE AS NITROGEN SOURCE (AEROBIC)
•
STUDIES ON THE PHOTODEGRADATION OF ANILINE
•
STUDIES ON THE PHOTOBIODEGRADATION OF ANILINE AS THE SOLE
CARBON AND NITROGEN SOURCE (ANAEROBIC)
•
STUDIES ON THE PHOTOBIODEGRADATION OF ANILINE AS THE SOLE
NITROGEN SOURCE (AEROBIC)
•
PERCENTAGE OF ANILINE DEGRADATION
•
QUANTIFICATION OF INDOLE
•
HPLC ANALYSIS
RESULTS AND DISCUSSION
• IN SILICO
•
PERCENTAGE OF XENOBIOTIC DEGRADING GENES
In Rhodopseudomonas palustris CGA009 = 15.92%
In Rhodobacter sphaeroides DSM 158
= 9.37%
In Rhodospirillum rubrum ATCC 11170 = 9.89%
CONT..
• GLOBAL MAP OF ENTIRE METABOLISM
FOR Rhodopseudomonas palustris CGA 009
CONT..
• FOR Rhodobacter sphaeroides DSM 158
CONT..
• FOR Rhodospirillum rubrum ATCC 11170
CONT..
• XENOBIOTIC DEGRADATION PATHWAYS
KEGG
- Total number of available annotated pathways for,
Rhodopseudomonas palustris CGA009 = 19
Rhodobacter sphaeroides DSM 158
= 20
Rhodospirillum rubrum ATCC 11170 = 12
METACYC
- Total number of available annotated pathways for,
Rhodopseudomonas palustris CGA009 = 32
Rhodobacter sphaeroides DSM 158
= 19
Rhodospirillum rubrum ATCC 11170 = 13
CONT..
•
SEQUENCE COMPARISON OF XENOBIOTIC DEGRADING GENES
CONT..
• PUTATIVE ENZYMES OF RHODOBACTER SPHAEROIDES DSM
158
PATHWAYS- 8
SUBSTRATE- 44
PRODUCT- 44
PUTATIVE ENZYMES- 44
CONT..
• SELECTION OF SUBSTRATE AND PATHWAY FOR
DEGRADATION
• MINING OF NAPHTHALENE AND ANTHRACENE
DEGRADATION
CONT..
•
INFORMATION ABOUT PUTATIVE FATTY ACID BETA HYDROXYLASE
(CYTOCHROME P450) (EC: 1.14.-.-)
CONT..
• SEQUENCE SIMILARITY OF PUTATIVE FATTY ACID BETA
HYDROXYLASE (CYTOCHROME P450) (EC: 1.14.-.-) RSP_2378
WITH OTHER TWO STRAINS
- NO SIMILARITY
- BUT SHOWS SIMILARITY OF ~ 85 TO 98% WITH OTHER
STRAINS OF RHODOBACTER SPHAEROIDES.
IN VIVO
• GROWTH OF RHODOBACTER SPHAEORIDES DSM 158
Table : Growth of Rhodobacter sphaeroides DSM 158 with all supplements
CONT..
• EFFECT OF ANILINE ON GROWTH OF RHODOBACTER
SPHAEROIDES DSM 158
Fig : Growth of Rhodobacter sphaeroides DSM 158 in the presence of Aniline
as an additional supplement at 0.5 and 1.0mM concentration.
CONT..
• GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN
PRESENCE OF ANILINE AS SOLE CARBON SOURCE
(ANAEROBIC)
Fig : Growth of Rhodobacter sphaeroides DSM 158 in presence of Aniline as
sole carbon source at 0.5mM and 1.0mM concentration.
CONT..
• GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN
PRESENCE OF ANILINE AS SOLE NITROGEN SOURCE
(ANAEROBIC)
Fig : Growth of Rhodobacter sphaeroides DSM 158 in presence of Aniline as
sole nitrogen source at 0.5mM and 1.0mM concentration.
CONT..
• GROWTH OF RHODOBACTER SPHAEROIDES DSM 158 IN
PRESENCE OF ANILINE AS NITROGEN SOURCE (AEROBIC)
CONT..
WHOLE CELL ABSOPRTION SPECTRUM
•
Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is
grown with Pyruvate as carbon source and NH4Cl as nitrogen source.
CONT..
Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is
grown with Aniline as carbon source and NH4Cl as nitrogen source at 0.5mM concentration
Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is
grown with Aniline as carbon source and NH4Cl as nitrogen source at 1.0mM
concentration
CONT..
Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is
grown with pyruvate as carbon source and Aniline as nitrogen source at 0.5mM concentration
Fig : The whole cell absorption spectrum of Rhodobacter sphaeroides DSM 158 which is
grown with pyruvate as carbon source and Aniline as nitrogen source at 1.0mM
concentration
SPECTROSCOPIC ANALYSIS OF ANILINE
DEGRADATION
CONT..
Absorption spectrum of culture supernatants
• Initial absorbance of
Aniline (Carbon source)
at 0.5mM concentration
• Overlay of 24, 48, 72
hours absorbance of
Aniline (Carbon source)
at 0.5mM concentration
• Final (96 hours)
absorbance of Aniline
(Carbon source) at
0.5mM concentration
Absorption spectrum of culture supernatants.. CONT..
• Initial absorbance of
Aniline (Carbon source)
at 1.0mM concentration
• Overlay of 24, 48, 72
hours absorbance of
Aniline (Carbon source)
at 1.0mM concentration
• Final (96 hours)
absorbance of Aniline
(Carbon source) at
1.0mM concentration
Absorption spectrum of culture supernatants.. CONT..
• Initial absorbance of
Aniline (nitrogen source)
at 0.5mM concentration
• Overlay of 24, 48, 72
hours absorbance of
Aniline (nitrogen source)
at 0.5mM concentration
• Final (96 hours)
absorbance of Aniline
(nitrogen source) at
0.5mM concentration
Absorption spectrum of culture supernatants.. CONT..
• Initial absorbance of
Aniline (nitrogen source)
at 1.0mM concentration.
• Overlay of 24, 48, 72
hours absorbance of
Aniline (nitrogen source)
at 1.0mM concentration.
• Final (96 hours)
absorbance of Aniline
(nitrogen source) at
1.0mM concentration.
• PHOTO BIODEGRADATION OF ANILINE BY RHODOBACTER
SPHAEROIDES DSM 158 UNDER AEROBIC DARK CONDITIONS
• QUANTIFICATION OF INDOLE
Culture supernatants were collected and added with double the
amount of freshly prepared salpers reagent. The absorbance was read at 535
nm against reagent blank.
From the standard graph, 0.1 OD of Absorbance = 13µg/ml of total indole.
Amount of total indole produced at 0.5mM concentration of Aniline = 2.6µg/ml
Amount of total indole produced at 1.0mM concentration of Aniline = 5.72µg/ml
PERCENTAGE OF ANILINE DEGRADATION
• Degradation of Aniline (as carbon source)
at 0.5mM concentration
= 29.06 %
• Degradation of Aniline (as carbon source)
at 1.0mM concentration
= 19.38%
• Degradation of Aniline (as nitrogen source)
at 0.5mM concentration
= 21.88%
• Degradation of Aniline (as nitrogen source)
at 1.0mM concentration
= 6.08%
HPLC ANALYSIS FOR PHOTOBIODEGRADATION OF
NITROBENZENE BY RHODOBACTER SPHAEROIDES DSM 158
Rhodobacter sphaeroides DSM 158
Centrifugation at 10,000 rpm for 15 minutes.
Culture supernatant
Ethyl Acetate Extraction (Thrice)
Separate organic layer
Condensation by Flash Rotary evaporator
After
Dryness
Redissolved in methanol (1 ml)
Filtration
Injection sample 20 μL
PHOTO BIODEGRADATION OF ANILINE
Fig: HPLC chromatogram of culture supernatants of Rhodobacter sphaeroides DSM 158
grown under anaerobic dark condition with Aniline as nitrogen source
PHOTO BIOTRANSFORMATION OF ANILINE
Fig: HPLC chromatogram of culture supernatant of Rhodobacter sphaeroides DSM 158
grown under anaerobic dark condition with Aniline as nitrogen source
SIMILAR PEAKS FORMED BETWEEN CONTROL AND PHOTO
BIODEGRADATION FINAL
DISSIMILAR PEAKS FORMED BETWEEN CONTROL
AND PHOTO BIODEGRADATION FINAL
COMPARISON OF SIMILAR PEAKS WITH PHOTO
DEGRADATION FINAL
IDENTIFIED PEAKS
UNIDENTIFIED PEAKS
FINAL RESULTS OF HPLC ANALYSIS
Fig : HPLC chromatograms showing formation of new intermediates or compounds.
The peaks represented by boxes were metabolites formed unique, obtained only with the
presence of Aniline and with the presence of the strain. The peaks represented by pink color
boxes are identified peaks (Table). The boxes represented by blue color are unidentified
peaks.
CONCLUSION
•
Novel study of INTEGRATING IN SILICO AND IN VIVO APPROACHES for
knowing the xenobiotic degradative capability of the strains
•
Through in silico approaches the hierarchy of xenobiotic degradation
capability of the strain was concluded as
Rhodopseudomonas palustris CGA 009
Rhodobacter sphaeroides DSM 158
Rhodospirillum rubrum ATCC 11170.
•
Use of in silico approaches SAVED LOTS OF TIME without wasting in doing
trial and error experiments
•
KNOWLEDGE BASED SUBSTRATE SELECTION through metabolic
databases was demonstrated than blind selection of the substrate
•
To prove the presence or EXPRESSION OF THE GENE RSP 2378 coding for
putative fatty acid beta hydroxylase for aniline degradation was initiated with
microbiological and metabolomics study
Cont..
• Though aniline was NOT TOXIC to growth of rhodobacter sphaeroides
dsm 158, but it did not support the growth of strain. hence it cannot
serve as either as CARBON SOURCE OR NITROGEN SOURCE
• It was reflected in the PRODUCTION OF BACTERIOCHOLROPHYLL. it
was heavily affected when aniline used as carbon or nitrogen source at
0.5mm and 1.0mm concentration. the use of lesser concentration of
aniline starting from 0.1mm concentration to minimum lethal
concentration for degradation study might help in knowing extensively
the degradative capability of the strain
• The DISAPPEARANCE OF ANILINE measured using uv
spectrophotometer RAISED CHAOS in concluding the percentage of
degradation which was solved to some extent USING HPLC analysis.
The COMPLETE DISAPPEARANCE OF ANILINE was observed
when used as nitrogen source at 0.5mm concentration. it was
concluded that the strain utilized aniline as nitrogen source.
Cont..
• The production of INDOLE AND ITS DERIVATIVES even at 0.5mm
concentration of aniline were observed, INDOLE 3 ALDEHYDE and
ANTHRANILIC ACID was identified among the metabolites formed.
it raises question whether aniline transformed to indole or aniline only
induced indole production, which was still unrevealed by our research
group
 There were SIX PEAKS observed, found UNIDENTIFIED which
might be the transformed products from aniline. further identification
of those metabolites using polychem analysis such as MS, FTIR, NMR
TECHNIQUES can be done in future.
 Further GENOMICS AND PROTEOMICS STUDY can be carried out
to report the degradation or transformation of aniline by the strain
RHODOBACTER SPHAEROIDES DSM 158.
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47. A. V. Chalam, C. Sasikala, C. V. Ramana, N. R. Uma, P. R. Rao Effect of Pesticides
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48. N. Rajasekhar, Ch. Sasikala, Ch. V. Ramana Toxicity of N-Containing Heterocyclic
Aromatic Compounds and their utilization for Growth by a Few Purple NonSulfur Bacteria Contam. Toxicol. (2000) 65:375–382