Function-Driven Metagenomics Jo Handelsman
Dec 16, 2015
Function-Driven Metagenomics
Jo Handelsman
What is Function-Driven Metagenomics?
•Clone DNA directly from environmental sample
•Screen clones for expressed functions
Advantage:Not limited by sequence recognition
Points to Consider About Metagenomics
• Genes in environment have relevance to human health
• Model systems necessary to develop ecological principles governing the human microbiome
Function-Driven Metagenomics
• Approach
• Example #1: Antibiotic resistance genes in soil
• Example #2: Insect gut microbiome
Collect soil
Extract DNA
Digest
Metagenomic library construction
Ligate into vector
Transform E. coli
Screen transformants
Example #1 Antibiotic Resistance Genes in Soil• Antibiotic resistance is a significant and
growing problem
• Where do resistance genes originate?
Goal:Identify the source, diversity, and
movement of antibiotic resistance genes in the environment
Why Soil?
• Most antibiotics originate from soil bacteria
• Most antibiotic producers carry resistance genes
• Other soil organisms must contend with antibiotics
• Soil is the richest microbial environment we know of (5,000-10,000 species; enormous strain variation)
6’-N-acetyltranferase
Aminoglycoside modification sites
Adapted from Kotra et al. 2000. Antimicrobial Agents & Chemotherapy
Nodes
Probability >0.90
Probability between 0.80 and 0.90
Probability between0.70 and 0.80
Probability between 0.50 and 0.70
unmarked
Phylogenetic analysis of aminoglycoside resistance genes (acetyltransferases)
Resistance genes from soil form a new clade of acetyltransferases.
NH
N
O
S
O OH
R2
A1) R1=
NH2
O
R2= HA3) R1= H2N
N
S
NO
R2=
O
R1
A2) R1=
OHR2=
NN
NN S
OH
NO
R
OOH
R=
HN NH
S
A) B)
NO
HN
OR
S
O
OH
R= R=
R=
A) C)
D)
NH2
OH
B) R=HO
NH2
E) R= O
N
N
O
O
N
O
Penicillin core A. Penicillin G. B-E, semi-synthetic β-lactam antibiotics. B. Amoxicillin. C. Ampicillin. D. Carbenicillin. E. Piperacillin.
Carbapenem coreB. R = imipenem.
Cephalosporin coreA1. Cephalexin. A2. Cefamandole. A3. Cefotaxime.
-Lactam antibiotics
Enzymatic Inactivation of -lactams
Classes of -lactamases- A, C, and D = serine β-lactamases
- B = metallo-β-lactamases
NO
HN
OR
S
O
OH
R= R=
R=
A) C)
D)
NH2
OH
B) R=HO
NH2
E) R= O
N
N
O
O
N
O
NH
N
O
S
O OH
R2
A1) R1=
NH2
O
R2= HA3) R1= H2N
N
S
NO
R2=
O
R1
A2) R1=
OHR2=
NN
NN S
OH
NO
R
OOH
R=
HN NH
S
A) B)
Penicillin core Cephalosporin core
Examples of-lactam Resistance in Soil Metagenome
Clone Insert Size
Resistance Resistance Gene Similarity
βLR1 6.1 ampicillin cephalothin
67% identity to
class A serine β-lactamase
Burkholderia
βLR2 12.1 amoxicillin
45% identity to
metallo β-lactamase
Janthinobacterium
βLR3 10.1 ceftazidime
48% identity to
class B β-lactamase
Chryseobacterium
βLR16 8.0
carbenicillin
(24o C)33% identity to
membrane protein Magnetospirillum
Metagenomics and Antibiotic Resistance
• Functional screening identified resistance genes that would not have been recognized by sequence alone
• Soil contains new clades of genes for resistance to aminoglycosides and β-lactams
• Do these genes move from soil to clinical settings?
• Do agricultural practices such as spraying apple trees with streptomycin affect the frequency of antibiotic resistance genes in soil?
Example #2Gypsy Moth
Ecological Questions
• What is the basis for robustness of a gut community?
• How does interspecies microbial signaling affect robustness?
Gypsy moth gut contains a simple model community with 5-10 species
Dissect larvae, separate bacteria
Extract DNA
Digest
Metagenomic library construction
Ligate into vector
Transform E. coli
Screen transformants
Search for Community Signals
• Screen clones for ability to induce quorum sensing
• Quorum sensing is bacterial communication
• Usually mediated by acylated homoserine lactones
500 bp
PROBABLE OXIDOREDUCTASE PROTEIN
PUTATIVE OXYGENASE SUBUNIT PROTEIN
CONSERVED HYPOTHETICAL PROTEIN
CONSERVED HYPOTHETICAL PROTEIN
PUTATIVE TRANSMEMBRANE PROTEIN
69.7%78.0%
Ralstonia solanacearum
GMI1000 genome 2547-7634 bp,
reversed
Gypsy moth metagenomic clone pBSS3
Gypsy Moth Midgut Quorum Sensing Inducing Clone
Putative oxygenase and conserved hypothetical protein required for production of signal
Scheme for Synthesis of Quorum Sensing Inducer
N
O
O
N
O
N
OH
N
O
OH
N
OH
O
Indoxyl
Isatin
O2O2Dioxindole
2-hydroxyindoxyl
Insect Gut Signal
• Indoxyl respresents a new class of quorum sensing inducer
• Genes would not have been recognized by sequence analysis alone
• Signal affects community structure
• Impact of signal on community robustness are in progress
Thanks to….
Soil MetagenomeHeather Allen Luke MoeChristian ReisenfeldLynn Williamson Brad BorleeChanghui GuanJitsupang (Koy) Rodbumrer
NIH, NSF, and HHMI
Insect MicrobiomeNichole BroderickChanghui GuanBen Shen/Jinhua JuKen Raffa