Effect of substrate type on toluene biodegradation following a horizontal gene

transfer event in Escherichia coli DH5α

Kaoru Ikuma, Ruoting Pei, Claudia K. GunschDepartment of Civil & Environmental Engineering, Duke

University, Durham, NC

Horizontal gene transfer (HGT) as a means to improve degradation of contaminants by microbes

http://www.sciencemuseum.org.uk/on-line/genes/232.asp

http://en.wikipedia.org/wiki/Bacterial_conjugation

Vertical gene transfer

Horizontal gene transfer

Plasmids can transform cells that come in contact with donor cells (conjugation)

Conjugated cells will not keep the plasmid unless it confers an environmental advantage!

→ Conjugation events may readily occur but many do not result in a functional phenotype

http://en.wikipedia.org/wiki/Bacterial_conjugation

Conjugation of the TOL plasmid chosen as model of horizontal gene transfer

Velázquez et al., 2005

Parental Strain Recipient Strain(Pseudomonas putida BBC443) (E. coli DH5α)

rp

No fluorescence

signal.Fluorescence

signal.

Bacterial Chromosome

TOL plasmid

Repressor Proteinrp

Pseudomonas putida BBC443 harboring a TOL plasmid tagged with GFP and kanamycinresistance used as donors

Christensen, et al., 1998

E. coli DH5α cells were conjugated with the TOL plasmid using filter mating

Recipient cells (E. coliDH5α) on membrane filter

Filter incubated in the presence of donor cells (P. putida BBC443)

Donor cells washed offFilter incubated

on agar plate

E. coli transconjugantsharboring TOL plasmid

http://filebox.vt.edu/users/chagedor/biol_4684/mfstrep.html

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Drinking_Water/jpgs/Drinking_water.html

Transfer of TOL plasmid into E. coli was verified through fluorescence microscopy

E. coli transconjugant cells

Mixture of P. putida cells and E. coli transconjugant cells

Time (Hours)0 20 40 60 80 100

Tolu

ene

Con

cent

ratio

n (p

pm)

-20

0

20

40

60

80

100

Abiotic ControlE. coli ConjugateP. Putida

E. coli transconjugants harboring the TOL plasmid could not grow with toluene as sole carbon source

The effects of the addition of glucose as carbon source along with toluene was tested

Pre-grown E. colitransconjugant cells

Toluene degradation over time

Catechol-2,3-dioxygenase (C23O)

enzyme assay

Incubated in the presence of toluene and glucose

Cloning of TOL plasmid genes

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120 140 160

time (hr)

tolu

ene

conc

entr

atio

n (p

pm)

Abiotic E. coli + tol

E. coli + glc0.5 + tol E. coli + glc2 + tol

E. coli + glc5 + tol

The addition of glucose significantly increased the rate of toluene degradation in E. colitransconjugants

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120 140 160

time (hr)

tolu

ene

conc

entr

atio

n (p

pm)

Abiotic E. coli + tol

E. coli + glc0.5 + tol E. coli + glc2 + tol

E. coli + glc5 + tol P.putida

The addition of glucose significantly increased the rate of toluene degradation in E. colitransconjugants

PCR confirmed the presence of TOL plasmid genes in the E. coli transconjugant cells

xyl E xylF xylJ xylK

P. putida E. coli P. putida E. coliP. putida E. coli

P. putida E. coli

Possibility of mutations in these genes introduced during the conjugation process

→ Sequencing of xyl genes in progress

~900 bp

0

0.02

0.04

0.06

0.08

0.1

E. coli + toluene

E. coli + glucose (0.5 g/L) + toluene

E. coli + glucose (2 g/L) + toluene

E. coli + glucose (5 g/L) + toluene

E. coli (no toluene)

P. putida + toluene

C23

O a

ctiv

ity n

orm

aliz

ed to

bio

mas

s (/m

in)

Preliminary results show slight increase in TOL enzyme activity with glucose addition

Conclusions and future work

Conjugation of E. coli with the TOL plasmid did not result in a functional phenotype (significant toluene degradation capabilities)

Glucose addition increased toluene degradation in E. colitransconjugants

Reducing power?Cometabolism?Other mechanisms?

Difference in TOL plasmid gene expression (qRT-PCR) under different conditions

HGT of TOL plasmid into strains other than E. coli and in mixed cultures

GC content of recipient genome may play a significant role in transfer efficiency/functional phenotype (Sorek et al., 2007)

Acknowledgements

Department of Civil and Environmental Engineering, Duke University

Annie ChenShuyi WangSara Morey

Special thanks to Dr. Søren Molin(Denmark Technical University) for the kind donation of P. putida strain BBC443

TOL plasmid contains genes encoding proteins that convert toluene to TCA cycle intermediates

Velázquez, et al., 2005

Toluene degradation curve

-10

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120 140 160

time (hr)

tolu

ene

conc

entr

atio

n (p

pm)

Abiotic E. coli + glc0.5 + tol

E. coli + glc2 + tol E. coli + glc5 + tol

E. coli + for0.001 + tol E. coli + for0.1 + tol

E. coli + for1 + tol