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

BYKARTHIKEYAN.B.S

BI0807

UNDER THE GUIDANCE OFDR.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.DAssociate Professor,Bacterial Discovery Lab,Center for Environment,Institute of Science and Technology,Jawaharlal Nehru Technological University,Hyderabad.

Dr.Ch.Venkata Ramana, M.Sc.,Ph.DProfessor,Anoxygenic Phototrophic Bacterial Lab,Department of Plant science,School of Life Science,University of Hyderabad,Hyderabad.

BIOINFORMATICS

XENOBIOTIC DEGRADATION

METABOLOMICS

MICROBIOLOGY

COMPARATIVE GENOMICS

BACTERIAL DISCOVERY

LABORATORY

METABOLIC DATABASES

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 OUTWHOLE GENOMES OF APB (Rhodopseudonomanas palustris

CGA009, Rhodobacter sphaeroides DSM 158, Rhodospirillum rubrum ATCC

11170)

GENOME WIDE COMPARISONS

(IMG JGI)

SEQUENCE BASED COMPARISONS

(KEGG, METACYC)

% OF XENOBIOTIC DEGRADATION CODING GENES IN ALL THE

SELECTED STRAINS

COMPARISON BETWEEN SPECIES

% OF ANNOTATED GENES CODING FOR XENOBIOTIC DEGRADATION

RESULT

IN SILICO

IN SILICO RESULT

ACCORDING TO AVAILABILITY OF STRAIN AND CHEMICAL

SELECTION OF STRAIN AND SUBSTRATE

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

OPTIMUM CONCENTRATION SELECTED

DEGRADATION OF CHEMICAL AT OPTIMUM CONCENTRATION USING

HPLC

QUALITATIVE ANALYSIS OF PRODUCTS OF DEGRADATION THROUGH HPLC

ESTIMATION OF INDOLEIN VIVO

Cont..

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/mlAmount 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

Culture supernatant

Centrifugation at 10,000 rpm for 15 minutes.

Ethyl Acetate Extraction (Thrice)

Condensation by Flash Rotary evaporator

Separate organic layer

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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