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