Bioremediation & Microbial Diversity: Applications of Molecular Biological Tools in Studying Novel Physiological Traits
Suneel Arjun Chhatre Aug 11, 2009
Aug 11, 2009 S. A. Chhatre 2
Spelman College
Microbes: The Earth’s Engine
~4 Billions years Capable of exploiting a vast range of energy
sources and thriving in almost every habitat For 2 billion years microbes were the only form of
life (all the biochemistries of life evolved) Basic ecosystem processes; biogeochemical cycles
and food chains, vital & elegant relationship between themselves and higher organisms
Aug 11, 2009 S. A. Chhatre 3
Spelman College
Microbial Diversity Biodiversity as a source of innovation in biotechnology International Convention on Biological Diversity defines
genetic resources as “ genetic material of actual potential value”
Microbial Diversity as major resource for biotechnological products and processes Food Biotechnology Metabolites (amino acids, antibiotics,
biopharmaceuticals) Enzymes Environmental Biotechnology Biological Fuels
Aug 11, 2009 S. A. Chhatre 4
Spelman College
Why is Microbial Diversity Important?
Critical for the sustainability of life on earth, including recycling of elements, maintenance of climate, degradation of wastes
Expand the frontiers of knowledge about the limits and strategies of life
Largest untapped reservoir of biodiversity Key roles in conservation of higher organisms and
in restoration of degraded ecosystems
Aug 11, 2009 S. A. Chhatre 5
Spelman College
Tapping the Untapped?
Aug 11, 2009 S. A. Chhatre 6
Spelman College
Role of Carbon
When we study the chemistry of life, carbon is at the center of the action
Living things transform carbon-based compounds voraciously, and microbes, as Earth’s most prolific and earliest-evolved life forms, do so most avidly
Carbon cycle on Earth is largely dependent on microbiological processes, and biodegradation constitutes one-half of the carbon cycle
Aug 11, 2009 S. A. Chhatre 7
Spelman College
The Beginning of Biodegradation
As old as life itself Prebiotic soup of organic molecules that
served as the precurosr of the molecules, constituted first life (the ancestral cell)
They must also have served as the energy sources (self replication requires energy)
Aug 11, 2009 S. A. Chhatre 8
Spelman College
Explosion of life must have consumed most of the organic molecules in prebiotic soup during the 1st billion years of Earth in the sustainence of first life
At that point, the richest source of food for life was other forms of life
Aug 11, 2009 S. A. Chhatre 9
Spelman College
This continues today Microbes produce lipases, proteases,
cellulases and ligninases that decmpose living organisms or their remains after death
Photosynthesis was an important development on the earth’s surface that allowed much greater biomass production and hence generated more molecules to be biodegraded
Aug 11, 2009 S. A. Chhatre 10
Spelman College
Importance of Microbial Diversity
Microbes harbor the greatest biological diversity and play a more important role in maintaining global processes
Microbes have been around since the start of the life at least 3.6 billion years ago (macroscopic ~ 1 billion years)
Microbes reproduce, and thus evolve new traits faster than macroscopic organisms
Aug 11, 2009 S. A. Chhatre 11
Spelman College
Number of bacteria attached to your body exceeds the entire human population on earth
Approximately 5X1030 prokaryotes reside on earth
500,000 species of insects, termites have 1000 sp of bacteria
Total number of bacteria in domestic animals is close to 4X1024
Aug 11, 2009 S. A. Chhatre 12
Spelman College
Identifying Novel Microbial Catalysis by Enrichment Culture & Screening
One gram of soil contains 109 bacteria, perhaps 10,000 different types
Pioneered by Beijerinck & Winogradsky Selective cultivation of one or more bacterial strains
obtained from complex mixture such as soil, sludges, water etc.
The method relies on using a particular organic compound as the sole carbon source or, less frequently, as the N, S or P source
Aug 11, 2009 S. A. Chhatre 13
Spelman College
Case Studies:
Hydrocarbon degrading potential of microbes (Oil Spill Remediation)
Reductive Dehalogenation (Degradation of Pesticide, Pentachlorophenol) in Sphingobium cholorophenolicum
Sulfur Oxidation Reactions & Acid Tolerance Resposnse in Halothiobacillus neapoitanus
Aug 11, 2009 S. A. Chhatre 14
Spelman College
Bioremediation of Oil Spills
EPA (2006): world wide consumption of petroleum was 84,979,000 barrels/day
Transportation Oil Spills Disasters
Torey Canyon 1967 (38 million gallons) Exxon Valdez 1989 (10 millions gallons plus) Westchester 2000 (567,000 gallons) Hurricane Katrina 2005 (7 millions)
Aug 11, 2009 S. A. Chhatre 15
Spelman College
Two Step Treatment Protocol
Containment: Step one is skimming the crude oil from the surface (Sawdust)
Mineralization: Step two is biodegradation of crude oil components by using bacterial catabolic properties (Consortium)
Aug 11, 2009 S. A. Chhatre 16
Spelman College
Goals
Enrichment, Isolation and Characterization of hydrocarbonoclastic microorganisms
Designing a consortium based on their catabolic properties and the composition of crude oil
Determination of efficacy of consortium for crude oil/hydrocarbon degradation
Osmotolerance (genetic manipulation)
Aug 11, 2009 S. A. Chhatre 17
Spelman College
Fig.4.1: COD Reduction in the Effluent of Oil-fed Semicontinuous Reactor
0
100
200
300
400
500
600
2 30 60 120 150 180
Time (Days)
CO
D (
mg
/L)
Enrichment of Bacteria
Oil Sludge: Semicontinuous batch reactor fed with crude oil for enrichment of hydrocarbon degraders
Serial Dilution & Plating (After six months when COD was 60% lowered)
Aug 11, 2009 S. A. Chhatre 18
Spelman College
Isolation & Characterization
Thirty five isolates Three Tier Screening
Primary: based on morphology, growth pattern, incubation time
Secondary: Antibiotic Sensitivity Tertiary: based on hydrocarbon degradation
potential (catechol, dodecane, tetracosane, eicosane, phenanthrene)
Aug 11, 2009 S. A. Chhatre 19
Spelman College
Designing the Consortium
Three of the isolates DSS6: Aliphatic degradation, biosurfactant GSS3: Aromatic degrader DSS8: Long chain aliphatic
Pseudomonas putida ATCC 102, known for consumption of down stream metabolites
Seed culture grown on catechol prior to crude oil degradation
Aug 11, 2009 S. A. Chhatre 20
Spelman College
Isolate DSS6: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 21
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Isolate DSS8: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 22
Spelman College
Isolate GSS3: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 23
Spelman College
Characterization of Isolates on the Basis of Catabolic
Pathway Using PCR Specific Primers based on the
catabolic properties PCR with total DNA of each isolate
and control dmpN-Phenol Hydroxylase Pseudomonas sp. (strain
CF600) xylE alkB
Aug 11, 2009 S. A. Chhatre 24
Spelman College
alkB-Alkane Hydroxylase
From OCT plasmid of Pseudomonas oleovorans
Aug 11, 2009 S. A. Chhatre 25
Spelman College
XylE-Catechol 2-3 Dioxygenase
Aug 11, 2009 S. A. Chhatre 26
Spelman College
Efficacy of Consortium for Biodegradation
Gas Chromatography Catechol grown consortium was applied to
degrade Crude Oil
A. Control
B. After 72 Hrs.
Aug 11, 2009 S. A. Chhatre 27
Spelman College
Gravimetric Analysis of Various Fractions
Figure 6.3: Utilization of Various Fractions of Crude Oil by the Designed Consortium
351
38.8 19
66.5
12.1 18
0
50
100
150
200
250
300
350
400
Aliphatic Aromatic Asphaltenes
Fractions of Crude Oil
Wei
gh
t in
mg
Control
Degraded
Figure 5.7: Growth of the designed consortium on Crude Oil
0
0.2
0.4
0.6
0.8
1
1.2
0 12 24 36 48 60 72 84
Time in Hours
Abs
orba
nce
at 6
20nm
Aug 11, 2009 S. A. Chhatre 28
Spelman College
Same methodology but crude oil was subjected to degradation individually
Alone, the efficiency was not as high as in group
Degradation by Individual Members
Gas Chromatograph of Crude Oil after 72 hrs
Aug 11, 2009 S. A. Chhatre 29
Spelman College
Biosurfactant Production by DSS6
Aug 11, 2009 S. A. Chhatre 30
Spelman College
Imparting Osmotolerance to Consortium
Pro ‘U’ operon (Dr. Gowrishankar, 1996)
Glycine-betaine uptake Subcloned in pMMB206 (a
broad host range vector) Growth in presence of 1M
Nacl Degradation of Model
Petroleum
Figure 7.4: Growth of NCC.DSS6 in LB medium amended with various concentrations of NaCl
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.5 0.6 0.7 0.8 0.9 1
Concentration of NaCl (M)
Ab
sorb
ance
at
620n
m (
24
hrs
) DSS6 (Wild)
DSS6 (Transformed)
Figure 7.5: Growth of NCC.DSS8 in LB medium amended with various concentrations of NaCl
00.10.2
0.30.40.50.6
0.70.80.9
0.5 0.6 0.7 0.8 0.9 1
Concentration of NaCl (M)
Ab
sorb
ance
at
620n
m
(24
hrs
)
DSS8 (Wild)
DSS8 (Transformed)
Aug 11, 2009 S. A. Chhatre 31
Spelman College
Model Petroleum Homogenous mixture of
representative hydrocarbons
1-dodecane (C12) 2-naphthalene (Dicyclic) 3-pentadecane (C15) 4-hexadecane (C16) 5-pristane (IS) 6-dibenzothiophene (Hetero) 7-phenanthrene (Tricyclic) 8-eicosane (C20) 9-tetracosane (C24) 10-octacosane (C28
Single peak with Capillary GC)
Aug 11, 2009 S. A. Chhatre 32
Spelman CollegePhysical Skimming of Crude Oil
Alkali Treated sawdust (high temp and pressure) Delignification causes increase in surface area Measured by Methylene Blue Isotherms, Mercury
Porosimetry; proved by Scanning Electron Microscopy
0
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300400
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600700
800900
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1200
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2400
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Methylene Blue concentration taken for experiment (ppm)
Met
hyle
ne B
lue
abso
rptio
n (p
pm)
Untreated
Treated
Aug 11, 2009 S. A. Chhatre 33
Spelman College
Physical Skimming of Crude Oil Crude Oil was spread over a trough
full of water Sprinkling of saw dust Skimming Gravimetric analysis proved ~ 90%
removal Cost effective and often necessary
Aug 11, 2009 S. A. Chhatre 34
Spelman College
Strategies to Study Evolutionary Origin of TCHQ dehalogenase in Sphingobium chlorophenolicum
Aug 11, 2009 S. A. Chhatre 35
Spelman College
What is TCHQ Dehalogenase? Reductive dehalogenase, removes 2 chlorine atoms in
the PCP degradation pathway in Sphingobium chlorophenolicum
PCP Degradation Pathway
Aug 11, 2009 S. A. Chhatre 36
Spelman College
Maleylacetoacetate Isomerase (MAAI):
Catalyzes the isomerization of maleylacetoacetate to fumaryl acetoacetate, a step in the degradation of Phenylalanine and Tyrosine
Aug 11, 2009 S. A. Chhatre 37
Spelman College
The sequence conservation in the active site regions of TCHQ dehalogenase and the known MAA isomerases
The ability of TCHQ dehalogenase to isomerize maleylacetone (MA), an analogue of MAA
The fact that both are members of zeta class of GST superfamily
The Relationship
Aug 11, 2009 S. A. Chhatre 38
Spelman College
Goals
Clone, sequence and express the MAA isomerase from S. chlorophenolicum and compare it with TCHQ dehalogenase
Determine what type of changes have occurred in order to enhance the dehalogenation reaction
In vitro evolution of maai into TCHQ dehalogenase Kinetic studies on dehalogenation
Aug 11, 2009 S. A. Chhatre 39
Spelman College
Experimental Approach I Knocking out maai gene in Pseudomonas
putida KT 2440
Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector)
Complementing the knockout mutant with genomic library and selecting on tyrosine
Preparation of plasmid from the colonies and sequencing
Aug 11, 2009 S. A. Chhatre 40
Spelman College
Insertion of Kanamycin Gene in the Middle of maai Gene
pBS + MAI with Kan
(pSS-MK)
Kan MAI/2MAI/1
Overlapping PCR
MAI+Kan
Aug 11, 2009 S. A. Chhatre 41
Spelman College
Genotype : PCR for kanamycin resistant gene inserted in
the middle of maai (bigger product)
Phenotype :
Growth on minimal media+Tyrosine as sole source of carbon
Growth on LB+kanamycin plates
Do We Have the Knockouts?
Aug 11, 2009 S. A. Chhatre 42
Spelman CollegePCR to verify maai gene with insertion of kanamycin resistance gene in mutants
With Long primers With short primers
M C6 C8 C9 C10 M C6 C8 C9 C10
Aug 11, 2009 S. A. Chhatre 43
Spelman College
Confirmation of knockouts by Phenotype
LB+Kan Tyrosine+Minimal Medium
Aug 11, 2009 S. A. Chhatre 44
Spelman College
Making a Genomic Library of S. chlorophenolicum
Optimization of partial digestion of genomic DNA with Sau 3A
Scale up the reaction with large quantity of DNA under right conditions
Digestion of Vector, dephosphorylation of digested vector
Ligation and electroporation
Aug 11, 2009 S. A. Chhatre 45
Spelman College
Optimization and Scale up of Partial Digestion Reaction
4Kb
~ 50 ug of genomic DNA was digested
Enzyme concentartion was .05 U/ug of DNA
Various enzyme concentration
Scale up with right concentration of Sau 3A
4Kb
Aug 11, 2009 S. A. Chhatre 46
Spelman College
4Kb
I V
Vector Preparation and Ligation
Broad host range vector pUCP-Nde (4Kb) BamHI digsetion & depshosphorylation with CIAP Ligation
Aug 11, 2009 S. A. Chhatre 47
Spelman College
Results ~ 28,000 clones
Restriction digestion profiles of some of the clones
4Kb
M U C U C U C U C U C U C M U C U C
M : MarkerU : UndigestedC : Cut (digested)
Aug 11, 2009 S. A. Chhatre 48
Spelman College
Complementation of Knockout with Full Copy of maai Gene
Cloning the entire gene (maai) in pUCP-Nde
Electroporation of the construct in electrocompetent knockout KT 2440 cells
4Kb
~ 650 bp
Colony PCR
Aug 11, 2009 S. A. Chhatre 49
Spelman College
Experimental Approach II
Degenerate PCR Amplification of unknown targets related to
multiple-aligned protein sequences 2 strategies :
1. Synthesize a pool of degenerate primers containing most or all possible nucleotides
2. Design single consensus primer across the highly conserved region
Aug 11, 2009 S. A. Chhatre 50
Spelman College
Primer Design for MAAI
Multiple Sequence Alignment (ClustalW) Block-Maker Codehop
A B C D
A
B
C
DSequences Blocks
Primers
Aug 11, 2009 S. A. Chhatre 51
Spelman College
Genomic Library in pSmart
sau3A digestion of G-DNA Ligation with pre-digested
vector
PCR Profile of Library Clones
Aug 11, 2009 S. A. Chhatre 52
Spelman College
Results : Degenerate PCR
Lane 1-7-14 : MarkerLane 2-8 : Sph. G-DNALane 3-9 : Sph. LibraryLane 4-10 : KT G-DNALane 5-11: PAO1Lane 6-12: KT ConstructLane 13 : Positive control
Aug 11, 2009 S. A. Chhatre 53
Spelman College
Site Directed Mutagenesis
Comparison of TCHQ dehalogenase sequence with known bacterial and eukaryotic MAAIs
Mutations in the active site region
Aug 11, 2009 S. A. Chhatre 54
Spelman College
Aug 11, 2009 S. A. Chhatre 55
Spelman College
Aug 11, 2009 S. A. Chhatre 56
Spelman College
Mutations in TCHQ dehalogenase
Aug 11, 2009 S. A. Chhatre 57
Spelman CollegeQuick-change Mutagenesis (Stratagene)
Aug 11, 2009 S. A. Chhatre 58
Spelman College
Sequencing
Aug 11, 2009 S. A. Chhatre 59
Spelman College
Deletion Mutant
PCR amplification of the 2 fragments with restriction sites Overlapping PCR for the full fragment Cloning in pET 21a
1 97 108 248
Aug 11, 2009 S. A. Chhatre 60
Spelman College
Results
PCR for 2 fragments
Overlapping PCR and vector
pET 21a
Product
Aug 11, 2009 S. A. Chhatre 61
Spelman College
Colony PCR and Sequencing
pcpC
Blast 2 with pcpC
Aug 11, 2009 S. A. Chhatre 62
Spelman College
Purification of TCHQ dehalogenase
Poor Yield (3mg/L) Tedious Prep Three different
columns Blue Agarose Mono-Q Superdex
Aug 11, 2009 S. A. Chhatre 63
Spelman College
Substrate Inhibition in TCHQ Dehalogenase
Third & Fourth steps Nucleophilic attack of
glutathione upon an electrophilic substrate to form a conjugate
MAAI & MPI isomerization of double bond, regenenerates glutathione
Reductive dehalogenation, 2 equiv of glutathione and results in oxidation to glutathione disulfide
Aug 11, 2009 S. A. Chhatre 64
Spelman College
Substrate Inhibition
The substrate primarily binds as TriCHQ- and is rapidly deprotonated to TriCHQ2- at the active site
TriCHQ2- is converted to it’s tautomer (TriCHQ* ) which is attacked by glutathione
Cys13 then attacks the glutathione conjugate, releasing the reduced product and forming a covalent bond between Cys13 and glutathione
Finally, the free enzyme is regenerated by thiol-disulfide exchange reaction with the second molecule of glutathione
It is profoundly inhibited by its aromatic substrates
Aug 11, 2009 S. A. Chhatre 65
Spelman College
Trade Off: Mutant I12A & I12S
Mutation of Isoleucine 12 to alanine or serine gives and enzyme which is not inhibited by the substrates
Weak binding of TriCHQ to ESSG Decrease in rate of dehalogenation
pH Dependent Protein Expression in Sulfur Oxidizing Bacteria
Aug 11, 2009 S. A. Chhatre 67
Spelman College
Deep Sea Thermal Vents
Temperatures as high as 404C Depths in 1000’s of meters Pressures >6000 psi Anoxic
Aug 11, 2009 S. A. Chhatre 68
Spelman College
Cold seeps
Temperatures 12 to 45 C pH 6.3 to 7.7 Salinity 1200 to 21000 S Eh -380 to -280 mV Depths in 100’s of m
Aug 11, 2009 S. A. Chhatre 69
Spelman College
Why study them?
Obvious interest in unique microbial physiologies Species thrive in the presence of high levels of toxic
compounds Marine and surface thermal vents long proposed as ‘source
of life’ Lateral gene transfer proposed as source for pathogenicity
in Proteobacteria Other adaptive responses in Proteobacteria species may
also have arisen from horizontal gene transfer Locations range from marine (Mid-Atlantic Ridge) to semi-
arid high altitude desert environments (Eddy Co., NM)
Aug 11, 2009 S. A. Chhatre 70
Spelman College
Model Organism
Halothiobacillus neapolitanus Isolated from a shallow marine vent Also found in cold seeps and municipal sewers Reduced and partially reduced inorganic sulfur
compounds as the sole source of energy Mildly halotolerant, mesophile pH range 8.5 to as low as 3.5
Aug 11, 2009 S. A. Chhatre 71
Spelman College
Our reasons to study H. neapolitanus
Wide pH range indicates potential for inducible acid tolerance response (ATR)
Chemolithoautotroph Proposed to use the ‘S4’ oxidation pathway Relationship to thermal vent species
Aug 11, 2009 S. A. Chhatre 72
Spelman College
“S4”Sulfur Oxidation Pathway
2SO3-2
2S0 2S-2
2SO4-2
1S2O3-2
2S2O3-2
S4O6-2
2AMP
2APS
2Pi
2ADP
2PPi
2ATP
2 H2O2 O2
4 H+
4 e-
6 H2O
12 H+, 8e-
6 H2O
12 H+, 12e-
? e-
2e-
2 H2O
4 H+, 4 e-
1
2 3 4 4
5
6
7
89
10
1 sulfide oxidase2 thiosulfate oxidase3 sulfur oxygenase4 sulfite reductase5 rhodanese ortetrathionate hydrolase6 TOMES7 adenyl sulfate reductase8 ADP sulfurylase9 ATP sulfurylase10 sulfite oxidase11 trithionate hydrolase
Sulfane sulfur ?
?
2S3O6-2
?
?
Aug 11, 2009 S. A. Chhatre 73
Spelman College
Plan of Action
Identify sulfur oxidizing activities Establish a baseline for physiology and protein
expression Determine pH dependence of physiology and
protein expression Establish correlations between physiological
changes and expression of individual genes
Aug 11, 2009 S. A. Chhatre 74
Spelman College
time0 10 20 30 40 50
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0Thiosulfate Growth Curve
Legend
pHthiosulfate
cfu/mltetrathionate
Aug 11, 2009 S. A. Chhatre 75
Spelman College
Time (sec)
0 10 20 30 40 50 60 70 80 90
% s
atur
atio
n
40
50
60
70
80
90
100
Sulfide Dependent Oxygen Consumption
Legend
Sulfide 100nmol
Sulfide 50nmol
0.1mM cyanide
0.1mM azide
1.0mM cyanide
1.0mM azide
ethanol blank
1.0mM rotenone
1.0mM Antimycin A
Aug 11, 2009 S. A. Chhatre 76
Spelman College
Time (sec)
0 10 20 30 40 50 60 70 80 90 100 110 120
% s
atur
atio
n
40
50
60
70
80
90
100
Thiosulfate Dependent Oxygen Consumption
Legend
THIOSULFATE AVE
five a
CN
N3
Anti A
Anti A etoh
Rotenone
Myxothiazol
Aug 11, 2009 S. A. Chhatre 77
Spelman CollegeSubstrate dependent oxygen consumption by Halothiobacillus neapolitanus
Aug 11, 2009 S. A. Chhatre 78
Spelman CollegeSubstrate dependent oxygen consumption by Halothiobacillus
neapolitanus Substrate amount Total O2 consumption O2 consumption rate
(nmol) (nmol) (nmolmin-1mg-1)
S-2 0 0 050 1727 2209100 33615 2147
S0* 0 0 050 8411 558100 17414 596
S2O3-20 0 0
50 997 786100 1925 825
S4O6-20 0 0
50 1556 786100 3159 825
S306-2 100 0.0 0.0
S5O6-2100 0.0 0.0
SO3-2 100 0.0 0.0
SO4-2 100 0.0 0.0
Aug 11, 2009 S. A. Chhatre 79
Spelman CollegeEffect of inhibitors on total O2 consumption*
Inhibitor S-2 S0 S2O3-2 S4O6
-2 Rotenone 817.8 934.4 100.4 5.7 97.6 6.3antimycin A 100.511.3 98.1 2.4 85 3.8 100.1 2.7TTFA 90.14 92 1.9 94.8 3.7 90.3 3.3myxothiazol 92.24.3 94.7 1.6 99.9 3.8 89.7 6.7NEM 21.34.5 16.7 4.0 3.9 8.4 23.7 4.9azide, 0.01mM1004 1002.2 1005 1003.9azide, 1mM 84.83.3 84.7 3.1 81.9 6.2 89.6 6.6cyanide, 0.01mM 1002.2 10017 1009 1006.2cyanide, 1mM 89.810.4 91.1 7.7 90.0 6.9 92.7 8.9
*values are expressed as percentage of control without inhibitor and without correction for changes in gas solubility due to inhibitors
Aug 11, 2009 S. A. Chhatre 80
Spelman CollegeEffect of inhibitors on rate of O2 consumption
Inhibitor S-2 S0 S2O3-2 S4O6
-2 rotenone 54.77% 92 4.6 99.2 2.7 46 8.2antimycin A 80.85.6% 84 7.7 97.7 3.8 81.2
7.7TTFA 52.36.1% 55.4 6 100 0.8
88.8 2.7myxothiazol 79.47.9% 88.4 4.7 98.6 3.0 94.7
4.6NEM 9.81.4% 1.6 0.9 2.2 1.7 6.1
2.8azide, 0.01mM97.3 3.8 94.7 3.8 99.2 1.8 97.8 6.7azide, 1mM 283.3% 24.4 2.6 27 3.9 25.5 7.0cyanide, 0.01mM 99.1 2.9 97.2 5.5 98 4.7 97.4
3.0cyanide, 1mM 30.87.8% 33.2 2.6 31.7 6.8 34 2.3
Aug 11, 2009 S. A. Chhatre 81
Spelman College
Substrate:Oxygen Stoichiometry
Substrate mol O2/mol S mol
O2/mol e-
S-2 ~3.3:1 0.4:1S0 ~1.5:1 0.6:1S2O3
-2 ~2:1 0.8:1
S4O6-2 ~3:1 0.25:1
Aug 11, 2009 S. A. Chhatre 82
Spelman College
pH Dependent Protein Expression
pH 6.5 pH 4.5
Aug 11, 2009 S. A. Chhatre 83
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Summary of Physiology at pH 7
Unique electron transport system – terminal oxidase is cyanide insensitive
Stoichiometry is not clear Change in expression profile at low pH (ATR)
Aug 11, 2009 S. A. Chhatre 84
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Purification of Proteins involved in Sulfur Oxidation
Aug 11, 2009 S. A. Chhatre 85
Spelman College
Cloning & Characterization of Genes N-terminus sequencing of proteins (C-554, C-549,
Thiosulfate Oxidase) Primer Designing Genomic Libraries
3-4 Kb 30-40 Kb
PCR Cloning & Sequencing Activity Assay, Spectrum
Aug 11, 2009 S. A. Chhatre 86
Spelman College
C-554
Wavelength (nm)
500 505 510 515 520 525 530 535 540 545 550 555 560 565 570 575 580 585 590 595 600
Abs
orba
nce
0.300
0.325
0.350
0.375
0.4000.400
0.425
0.450
0.475
0.500
0.525
0.550
0.575
0.600
Legendcells grown at pH 4.5
cells grown at pH 6.5
Aug 11, 2009 S. A. Chhatre 87
Spelman College
C-554
Wavelength (nm)
500 510 520 530 540 550 560 570 580 590 600
Abs
orba
nce
0.150
0.175
0.200
0.225
0.250
0.275
0.3000.300
0.325
0.350
0.375
0.4000.400
0.425
0.4500.450
0.475
0.500
Legendcells grown at pH 4.5
cells grown at pH 6.5
Aug 11, 2009 S. A. Chhatre 88
Spelman College
Future Directions
Quenched oxygen consumption assays Measure NAD(P)/NAD(P)H ratios Measure P/O ratios pH dependence Identification of genes and gene products Real time PCR to verify changes in expression ‘Knock-out’ mutants
Aug 11, 2009 S. A. Chhatre 89
Spelman College
Acknowledgements
National Environmental Engineering Research Institute (NEERI)/ Indian Institute of Technology, Roorkee, India Department of Biotechnology (DBT)
MCDB/Chemistry, University of Colorado at Boulder, CO NIH, NSF, DOE
Chemistry, Eastern New Mexico University, Portales, NM NIH NCRR P20-61480
Aug 11, 2009 S. A. Chhatre 90
Spelman College
Acknowledgements Students
Anton Iliuk Ben Goldbaum Eliseo Castillo John Latham Joaquin DeLeon Nalini Anamula Ramu Kakumanu Neela Gamini
Dr. Suneel Chhatre
Collaborators Sabine Heinhorst Gordon Cannon
NIH NCRR P20-61480ENMU
Aug 11, 2009 S. A. Chhatre 91
Spelman CollegeFuture Directions
Purification of MAAI from Sphingobium Characterization of TCHQ dehalogenase
mutants
Aug 11, 2009 S. A. Chhatre 92
Spelman College
Acknowledgements
Copley Lab
Gill lab
Aug 11, 2009 S. A. Chhatre 93
Spelman College
CODEHOP (Consensus-degenerate Hybrid Oligonucletide Primers)
Short 3’ degenerate core and a 5’non-degenerate consensus clamp
Reducing the length 3’ core decreases the total number of primers
Hybridization of the 3’ degenerate core with template is stabilized by non-deg 5’ clamp
Aug 11, 2009 S. A. Chhatre 94
Spelman College
CODEHOP ….
Aug 11, 2009 S. A. Chhatre 95
Spelman College
CODEHOP Output
Aug 11, 2009 S. A. Chhatre 96
Spelman College
Amplification of maai from Pseudomonas
Primer design based on only two maai sequences, KT2440 and PAO1
G-DNA as a template Results of combination of
Forward 1 primer with 3 different reverse primers
Aug 11, 2009 S. A. Chhatre 97
Spelman CollegeQuick-change Mutagenesis (Stratagene)
Aug 11, 2009 S. A. Chhatre 98
Spelman CollegeFuture Directions
Genomic Library in pBBR1tp Purification of Homogentisate dioxygenase Characterization of TCHQ dehalogenase
mutants
Aug 11, 2009 S. A. Chhatre 99
Spelman College
Experimental approach
Knocking out maai gene in Pseudomonas putida KT 2440
Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector)
Complementing the knockout mutant with genomic library and selecting on tyrosine
Preparation of plasmid from the colonies and sequencing
Aug 11, 2009 S. A. Chhatre 100
Spelman College
Creation of Knockout Mutant by Homologous Recombination
Mark
er
pK
nock
-Km
Tru
ncated
maai
maai from
KT
2440
pKnock System
Aug 11, 2009 S. A. Chhatre 101
Spelman CollegeInsertion of Kanamycin Gene in the Middle of maai Gene
pBS + MAI with Kan
(pSS-MK)
Kan MAI/2MAI/1
Overlapping PCR
MAI+Kan
Aug 11, 2009 S. A. Chhatre 102
Spelman CollegePCRs
maai 1 maai 2
~350 bp
Overlapping PCR
~ 1.6 Kb
Kan gene
~ 800 bp
Aug 11, 2009 S. A. Chhatre 103
Spelman CollegeConstruction of pSS-MK
Gel extraction of right size fragment from PCR
Digestion with Hind III and BamH1
Ligation Electropration in XL1Blue
cells Plating on LB+Kan and
LB+ Amp media
M V I
3 Kb
1.6 Kb
Aug 11, 2009 S. A. Chhatre 104
Spelman CollegeExpression of Kan Gene in Knockouts
No growth on LB+Kan plates
Several colonies on LB+Amp Plates
3 Kb
1.6 Kb
Complete Gene Primers
ATG GAG CTG TAC ACC TAT TAC CGT TCC ACC TCG --- --- --- --- --- GCC ATC ATT GGT TGC GAC ATT CAT ATG ATT GAA CAA GAT GGA TTG CAC GCA GGT TCT --- --- --- ---
Incomplete Gene Primers (-25 bases)
CCA CCT CGT CCT ACC GGG TGC GCA TTG CCC --- --- --- --- --- --- --- CGG CCA TCA TTG GTT GCG ACA TTC ATA TGA TTG AAC A-------- ---
Aug 11, 2009 S. A. Chhatre 105
Spelman CollegeMaking Knockouts
Electroporation of the constructs and ligation mix into KT 2440 cells
4 colonies showed 1.6 Kb fragment
M C 1 2 3 4 5 6 7 8 9 10
1.6 Kb
Aug 11, 2009 S. A. Chhatre 106
Spelman College
Plasmid or Homologous Recombimation?
Lane 1-4 : Plasmid DNA prepLane 5 : uncut pBS vectorLane 6-9 : Genomic DNA prep
Looks like we have homologous recombination!
Aug 11, 2009 S. A. Chhatre 107
Spelman College
Making a Genomic Library of S. chlorophenolicum
Optimization of partial digestion of genomic DNA with Sau 3A
Scale up the reaction with large quantity of DNA under right conditions
Digestion of Vector, dephosphorylation of digested vector
Ligation and electroporation
Aug 11, 2009 S. A. Chhatre 108
Spelman College
Optimization and Scale up of Partial Digestion Reaction
4Kb
~ 50 ug of genomic DNA was digested
Enzyme concentartion was .05 U/ug of DNA
Various enzyme concentration
Scale up with right concentration of Sau 3A
4Kb
Aug 11, 2009 S. A. Chhatre 109
Spelman College
4Kb
I V
Vector Preparation and Ligation
Broad host range vector pUCP-Nde (4Kb)BamHI digsetion & depshosphorylation with CIAPLigation
Aug 11, 2009 S. A. Chhatre 110
Spelman College
Results
~ 28,000 clones
Restriction digestion profiles of some of the clones
4Kb
M U C U C U C U C U C U C M U C U C
M : MarkerU : UndigestedC : Cut (digested)
Aug 11, 2009 S. A. Chhatre 111
Spelman CollegeComplementation of Knockout with Full Copy of maai Gene
Cloning the entire gene (maai) in pUCP-Nde
Electroporation of the construct in electrocompetent KT 2440 cells
4Kb
~ 650 bp
Colony PCR
Aug 11, 2009 S. A. Chhatre 112
Spelman College
Summary
Obtain a real knockout (not the contaminant)
Ligation reaction
Positive control with endogenous promoter
Aug 11, 2009 S. A. Chhatre 113
Spelman College
Experimental Approach I
Knocking out maai gene in Pseudomonas putida KT 2440
Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector)
Complementing the knockout mutant with genomic library and selecting on tyrosine
Preparation of plasmid from the colonies and sequencing
Aug 11, 2009 S. A. Chhatre 114
Spelman CollegeInsertion of Kanamycin Gene in the Middle of maai Gene
pBS + MAI with Kan
(pSS-MK)
Kan MAI/2MAI/1
Overlapping PCR
MAI+Kan
Aug 11, 2009 S. A. Chhatre 115
Spelman College
Optimization and Scale up of Partial Digestion Reaction
4Kb
~ 50 ug of genomic DNA was digested
Enzyme concentartion was .05 U/ug of DNA
Various enzyme concentration
Scale up with right concentration of Sau 3A
Aug 11, 2009 S. A. Chhatre 116
Spelman College
4Kb
I V
Vector Preparation and Ligation
Broad host range vector pUCP-Nde (4Kb)BamHI digsetion & depshosphorylation with CIAPLigation
No colonies!
Aug 11, 2009 S. A. Chhatre 117
Spelman CollegeComplementation of Knockout with Full Copy of maai Gene
Cloning the entire gene (maai) in pUCP-Nde, pTZ100, pBBR1tp
Electroporation of the construct in electrocompetent knockout KT 2440 cells
4Kb
~ 650 bp
Colony PCR
Aug 11, 2009 S. A. Chhatre 118
Spelman College
Site Directed Mutagenesis in TCHQ dehalogenase
Comparison of TCHQ dehalogenase sequence with human maleylacetoacetate isomerase
Aug 11, 2009 S. A. Chhatre 119
Spelman College
Mutations in TCHQ dehalogenase
Aug 11, 2009 S. A. Chhatre 120
Spelman College
Experimental Approach II
Glutathione agarose (N-linked) does bind TCHQ dehalogenase
Does MAAI (P.putida) binds to it?
Aug 11, 2009 S. A. Chhatre 121
Spelman College
Purification of MAAI from P. putida
P. putida MAAI with His tag (pET21a)
Wash 2
Wash 1
Flow
throu
ghC
rude
Elute
Aug 11, 2009 S. A. Chhatre 122
Spelman College
Purified MAAI on Glutathione Agarose
Elute
Wash
Flow
throu
ghL
oad
Aug 11, 2009 S. A. Chhatre 123
Spelman College
Sphingobium chlorophinolicum grown on Tyrosine
Wash
Flow
throu
ghSu
pernatent
Crud
e
Elutions
Aug 11, 2009 S. A. Chhatre 124
Spelman College
Experimental Approach III
Tyrosine degradation cassette in E. coli
Arias-Barran et al, J. Bac 2004
Aug 11, 2009 S. A. Chhatre 125
Spelman College
Colony PCR
M Mutants C
Aug 11, 2009 S. A. Chhatre 126
Spelman CollegeQuick-change Mutagenesis (Stratagene)
Aug 11, 2009 S. A. Chhatre 127
Spelman College
Isolate DSS6: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 128
Spelman College
Isolate DSS8: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 129
Spelman College
Isolate GSS3: Colony Characteristics
Aug 11, 2009 S. A. Chhatre 130
Spelman College
Aug 11, 2009 S. A. Chhatre 131
Spelman College
Aug 11, 2009 S. A. Chhatre 132
Spelman College
Aug 11, 2009 S. A. Chhatre 133
Spelman College
Aug 11, 2009 S. A. Chhatre 134
Spelman College
Bioremediation of Contaminated Soil