microbial diversity: tapping the untapped

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

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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


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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


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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


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Tapping the Untapped?


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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


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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)


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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


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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


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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


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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


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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


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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


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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)


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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)


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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)


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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)


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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)


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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


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Isolate DSS6: Colony Characteristics


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Isolate GSS3: Colony Characteristics


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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


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alkB-Alkane Hydroxylase

From OCT plasmid of Pseudomonas oleovorans


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XylE-Catechol 2-3 Dioxygenase


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Efficacy of Consortium for Biodegradation

Gas Chromatography Catechol grown consortium was applied to

degrade Crude Oil

A. Control

B. After 72 Hrs.


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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


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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


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Biosurfactant Production by DSS6


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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)


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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)


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

100200

300400

500

600700

800900

1000

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

Methylene Blue concentration taken for experiment (ppm)

Met

hyle

ne B

lue

abso

rptio

n (p

pm)

Untreated

Treated


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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


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Strategies to Study Evolutionary Origin of TCHQ dehalogenase in Sphingobium chlorophenolicum


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What is TCHQ Dehalogenase? Reductive dehalogenase, removes 2 chlorine atoms in

the PCP degradation pathway in Sphingobium chlorophenolicum

PCP Degradation Pathway


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Maleylacetoacetate Isomerase (MAAI):

Catalyzes the isomerization of maleylacetoacetate to fumaryl acetoacetate, a step in the degradation of Phenylalanine and Tyrosine


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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


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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


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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


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Insertion of Kanamycin Gene in the Middle of maai Gene

pBS + MAI with Kan

(pSS-MK)

Kan MAI/2MAI/1

Overlapping PCR

MAI+Kan


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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?


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With Long primers With short primers

M C6 C8 C9 C10 M C6 C8 C9 C10


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Confirmation of knockouts by Phenotype

LB+Kan Tyrosine+Minimal Medium


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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


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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


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4Kb

I V

Vector Preparation and Ligation

Broad host range vector pUCP-Nde (4Kb) BamHI digsetion & depshosphorylation with CIAP Ligation


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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)


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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


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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


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Primer Design for MAAI

Multiple Sequence Alignment (ClustalW) Block-Maker Codehop

A B C D

A

B

C

DSequences Blocks

Primers


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Genomic Library in pSmart

sau3A digestion of G-DNA Ligation with pre-digested

vector

PCR Profile of Library Clones


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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


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Site Directed Mutagenesis

Comparison of TCHQ dehalogenase sequence with known bacterial and eukaryotic MAAIs

Mutations in the active site region


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Mutations in TCHQ dehalogenase


Spelman CollegeQuick-change Mutagenesis (Stratagene)


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Sequencing


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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


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Results

PCR for 2 fragments

Overlapping PCR and vector

pET 21a

Product


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Colony PCR and Sequencing

pcpC

Blast 2 with pcpC


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Purification of TCHQ dehalogenase

Poor Yield (3mg/L) Tedious Prep Three different

columns Blue Agarose Mono-Q Superdex


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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


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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


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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


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Deep Sea Thermal Vents

Temperatures as high as 404C Depths in 1000’s of meters Pressures >6000 psi Anoxic


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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


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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)


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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


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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


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“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

?

?


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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


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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


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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


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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


Spelman CollegeSubstrate dependent oxygen consumption by Halothiobacillus neapolitanus


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


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


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


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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


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pH Dependent Protein Expression

pH 6.5 pH 4.5


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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)


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Purification of Proteins involved in Sulfur Oxidation


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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


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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


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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


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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


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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


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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


Spelman CollegeFuture Directions

Purification of MAAI from Sphingobium Characterization of TCHQ dehalogenase

mutants


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Acknowledgements

Copley Lab

Gill lab


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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


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CODEHOP ….


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CODEHOP Output


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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


Spelman CollegeFuture Directions

Genomic Library in pBBR1tp Purification of Homogentisate dioxygenase Characterization of TCHQ dehalogenase

mutants


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Experimental approach

Knocking out maai gene in Pseudomonas putida KT 2440





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Creation of Knockout Mutant by Homologous Recombination

Mark

er

pK

nock

-Km

Tru

ncated

maai

maai from

KT

2440

pKnock System


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


Spelman CollegePCRs

maai 1 maai 2

~350 bp

Overlapping PCR

~ 1.6 Kb

Kan gene

~ 800 bp


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


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-------- ---


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


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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!


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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


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4Kb





4Kb


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4Kb

I V


Broad host range vector pUCP-Nde (4Kb)BamHI digsetion & depshosphorylation with CIAPLigation


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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)


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


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Summary

Obtain a real knockout (not the contaminant)

Ligation reaction

Positive control with endogenous promoter


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Experimental Approach I

Knocking out maai gene in Pseudomonas putida KT 2440





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


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4Kb






Spelman College

4Kb

I V


Broad host range vector pUCP-Nde (4Kb)BamHI digsetion & depshosphorylation with CIAPLigation

No colonies!


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


Spelman College

Site Directed Mutagenesis in TCHQ dehalogenase

Comparison of TCHQ dehalogenase sequence with human maleylacetoacetate isomerase


Spelman College

Mutations in TCHQ dehalogenase


Spelman College

Experimental Approach II

Glutathione agarose (N-linked) does bind TCHQ dehalogenase

Does MAAI (P.putida) binds to it?


Spelman College

Purification of MAAI from P. putida

P. putida MAAI with His tag (pET21a)

Wash 2

Wash 1

Flow

throu

ghC

rude

Elute


Spelman College

Purified MAAI on Glutathione Agarose

Elute

Wash

Flow

throu

ghL

oad


Spelman College

Sphingobium chlorophinolicum grown on Tyrosine

Wash

Flow

throu

ghSu

pernatent

Crud

e

Elutions


Spelman College

Experimental Approach III

Tyrosine degradation cassette in E. coli

Arias-Barran et al, J. Bac 2004


Spelman College

Colony PCR

M Mutants C


Spelman College



Spelman College

Isolate GSS3: Colony Characteristics


Spelman College


Spelman College

Bioremediation of Contaminated Soil

microbial diversity: tapping the untapped

Health & Medicine

forms of life

form of life

sustainability of life

chemistry of life

explosion of life

years microbes

microbial diversity

earliestevolved life