accessing the uncultivable: unraveling activities from
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Accessing the Uncultivable: Unraveling Activities from Accessing the Uncultivable: Unraveling Activities from MetagenomicMetagenomic
Libraries Generated from Dry and Rain Libraries Generated from Dry and Rain
Forest Soils in Puerto Rico Using Functional and Forest Soils in Puerto Rico Using Functional and SequenceSequence--based Approachesbased Approaches
NCSS Conference at Las Cruces, NMNCSS Conference at Las Cruces, NM
JosJoséé
M. CruzM. Cruz--GarcGarcííaa11, Pedro J. Ondina, Pedro J. Ondina11, Manuel Ortega, Manuel Ortega11, , RosivetteRosivette
SantiagoSantiago11, , Jean C. CruzJean C. Cruz--HernHernáándezndez11, , AmarisAmaris
TorresTorres11, Francisco S, Francisco Sááncheznchez11, Orlando Soto, Orlando Soto--
CCááceresceres11, , LilliamLilliam
CasillasCasillas22, , andand
Carlos RCarlos Rííos Velos Veláázquezzquez33
University of Puerto Rico at University of Puerto Rico at MayagMayagüüezez, , 11Industrial Biotechnology Program and Industrial Biotechnology Program and 33Biology Department, Biology Department, 22University of Puerto Rico at University of Puerto Rico at HumacaoHumacao, Biology Department, Biology Department
May 11, 2009May 11, 2009
Soil: A valuable natural resourceSoil: A valuable natural resource
Why Why metagenomicsmetagenomics??
Few microorganisms can be cultivatedFew microorganisms can be cultivated
Access to uncultivable microorganismsAccess to uncultivable microorganisms
Why Why metagenomicsmetagenomics??
Bacterial Clone
Novel Activity
None
Antimicrobial agents
Biodegradation enzyme(s)
High temperature tolerance
Unraveling new activities with medical and/or Unraveling new activities with medical and/or biotechnological applicationsbiotechnological applications
Why Why metagenomicsmetagenomics??
1% 1% + 99%
Cultivating techniques Metagenomics
Why Why metagenomicsmetagenomics??
Monitoring: two approachesMonitoring: two approaches
Functional metagenomics Sequence‐based metagenomics
Expression
Transcription
Transduction
SecretionAATCGACCTAATTCTTAGCTGGATTAAG
Monitoring: two approachesMonitoring: two approaches
Functional metagenomics Sequence‐based metagenomics
•Microbial diversity
•Production of antimicrobial
agents
•Antibiotic resistance activity
•Production of antimicrobial
agents
•Detection of enzymatic activity
MetagenomicMetagenomic
library generationlibrary generation
MetagenomicMetagenomic
library generationlibrary generation
++
Genomic DNA extraction
Vector
Genomic DNA
Cultivable host
Ligation
Insert
Transformation
“Metagenomic Library”
Sampling SitesSampling Sites
http://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif
Sampling SitesSampling Sites
Guánica
Dry Forest Yunque
Rainforesthttp://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif
Sampling SitesSampling Sites
Guánica
Dry Forest Yunque
Rainforesthttp://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif
Antimicrobial activityAntimicrobial activity
Polyketide
Synthases
amplifications Overlay assays
Library clones
Assay strains
Top agar
Overlay
Zones of inhibition
A B
Detection of Detection of ureaseurease
using the using the ureaseurease testtest
Urease
test
NH2NH2
C
O
+ OH21. urease
OO C + NH32 C
O
O-
O-
NH4+
NH4+
2. H2O
(-) (+)
Antibiotic resistanceAntibiotic resistance
Fosmid
libraries screened for antibiotic resistance
ConclusionsConclusions
A total of 8 metagenomic libraries from two forests have been generated (~850,000 clones).There are differences in microbial diversity between the two forests.Through sequence-based analysis the presence of ampliconsassociated with production of antimicrobial agents has been detected.4 examples of functional analyses have been used: production of antimicrobial agents, and epoxide hydrolase, urease, and antibiotic resistance activity.Ongoing experiments involve lipase detection, transposonmutagenesis to identify genes associated with activity, and further sequencing and screening efforts.
AcknowledgementsAcknowledgements
Dr. Jo Handelsman, Lynn Williamson and fellow researchers at University of Wisconsin-MadisonGraduate and undergraduate students of the Microbial Biotechnology and BioprospectsLaboratory at UPRMMr. Dubiezel MedinaUSDA-CSREES 2007-02386PR-LSAMP UPRM
http://http://www.cohemis.uprm.edu/gemsprwww.cohemis.uprm.edu/gemspr//
The TEAMThe TEAM
Results –
Restriction digest analysis
IKB IKB
Vector7.5 kbp
A BRestriction digest analysis of (A) Guánica
dry forest library clones and (B) Yunque
rainforest library clones digested with NotI. The line indicates the fosmid
vector. Comparing the amount of internal NotI
recognition sites between both samples, sample(A) contained DNA inserts higher in GC content than sample (B).
NotI
recognition site 5’-GCGGCCGC-3’
Diversity analyses
16S rDNA
libraries TRFLP
Genomic DNA extraction
16S rDNAPCR amplification
TOPO TA cloning Digestion
EpoxideEpoxide
hydrolasehydrolase
detection using detection using the adrenaline testthe adrenaline test
OOH
OH
O
O
OH
N
CH3
I
O
O O-
ONa
+
I
O-
OO
Na+
+ OH2
3. back - titration
adrenochrome
adrenaline
OH
OH
NHCH3
OH
1. epoxide hydrolase
2. oxidation
Epoxide
hydrolase
activity: adrenaline test
(-)
(+)
Results -
Epoxide
hydrolase
detection
Example of adrenaline test assay for Guánica
dry forest library subpool
6. Odd rows indicate actual assay, even rows represent unassayed
duplicates. Sample (A) above was induced, (B) was not. The well for the last column in row 8 for sample (B) is Escherichia coli. Under these experimental conditions, it appears induction does not modify results significantly.
12345678
12345678
A
B
Results -
Antimicrobial activity detection
(A) Example of 20 screened plates, forK. pneumoniae.
(B) Example of 40 screened plates, 20 for S. aureus
and 20 for
K. pneumoniae.
TRFLP
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