Functional genomics of food-borne pathogens [Elektronische Ressource] / vorgelegt von Thilo M. Fuchs

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Functional genomics of food-borne pathogens Habilitationsschrift vorgelegt von Thilo M. Fuchs Lehrstuhl für Mikrobielle Ökologie Department für Biowissenschaftliche Grundlagen Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Freising 2006 Contents page 1. Summary 1 2. Introduction: functional genomics 2 3. Genome-wide insertional-duplication mutagenesis (IDM) 8 4. The essential genes set of Salmonella typhimurium 12 5. Intracellular replication of Listeria monocytogenes 19 6. Association of Yersinia enterocolitica with invertebrates 24 7. Acknowledgement 31 8. Bibliography 32 9. Author’s publications and patents 39 10. Curriculum vitae 42 11. Appendix: selected publications 44 Summary The growing number of complete microbial genome sequences and the ready availability of their annotation provide a powerful data base for studying the biology of microorganisms. In this work, two distinct high-throughput approaches are described to exploit genomics of pathogenic bacteria, insertional-duplication mutagenesis (IDM) and expression profiling using the luciferase reporter system.

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• Published by: technische_universitat_munchen
• Published: 01 January 2006
• Reads: 16
• Language: English

Exrait

Functional genomics

of food-borne pathogens

Habilitationsschrift

vorgelegt von

Thilo M.

F

u

c

h

s

Lehrstuhl für Mikrobielle Ökologie

Department für Biowissenschaftliche Grundlagen

Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,

Landnutzung und Umwelt

Freising

2006

Contents

page

1.

Summary

1

2.

Introduction: functional genomics

2

3.

Genome-wide insertional-duplication mutagenesis (IDM)

8

4.

The essential genes set of

Salmonella typhimurium

12

5.

Intracellular replication of

Listeria monocytogenes

19

6.

Association of

Yersinia enterocolitica

with invertebrates

24

7.

Acknowledgement

31

8.

Bibliography

32

9.

Author’s publications and patents

39

10.

Curriculum vitae

42

11. Appendix: selected publications

44

1

Summary

The growing number of complete microbial genome sequences and the ready availability

of their annotation provide a powerful data base for studying the biology of

microorganisms. In this work, two distinct high-throughput approaches are described

t

o

e

x

p

l

o

i

t

g

e

n

o

m

i

c

s

o

f

pathogenic bacteria,

i

n

s

e

r

t

i

o

n

a

l-duplication mutagenesis (IDM)

a

n

d

expression profiling using the luciferase reporter

s

y

s

t

e

m. Their genome-wide application

t

o

t

h

e

f

o

o

d-borne pathogens

Salmonella typhimurium

,

Listeria

m

o

n

o

c

y

t

o

g

e

n

e

s

a

n

d

Yersinia enterocolitica

led to new insights into the complex world of microbial life in terms

of (I) the minimal gene set, (II) intracellularly required factors, and (III) the association

with invertebrates.

I)

Minimalism

: The essential gene set of

S. typhimurium

was defined by a no

v

e

l

g

e

n

e

t

i

c

strategy. Small, randomly generated chromosomal fragments of this pathogen

w

e

r

e

cloned into a temperature-sensitive vector, and the resulting mutagenic library was

grown under permissive conditions. Upon switching to non-permissive temperature,

genes with essential functions under laboratory conditions could be trapped following

discrimination between lethal and non-lethal recombination events. Further

characterization of a total of ~500 fragments revealed 145 known essential genes and

112 functionally characterised or hypothetical genes not yet demonstrated to be essential

for a bacterial cell

;

t

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1

1

%

o

f

t

h

e

Salmonella

genome.

II)

Specialization

: More than

1,000 IDM mutants

o

f

t

h

e facultative intracellular

pathogen

L. monocytogenes

were screened for their phenotypes in human epithelial cells.

The genetic analysis of severely attenuated mutants revealed a huge number of listerial

g

e

n

e

s

required for replication in non-phagocytic cells, thus dissecting the genome of

Listeria

in

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r

m

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.

T

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acquisition of

species-specific genes and the usage of alternative sugar and nitrogen sources could be

demonstrated as novel strategies that enable this pathogen to survive in

t

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e

c

y

t

o

s

o

l

o

f

the host cell

.

III)

Associ

a

t

ion

: A promoter fusion library of

Y. enterocolitica

was constructed by the

transposon-mediated chromosomal insertion of the luciferase reporter

,

a

n

d

t

h

e

transcriptional response of

t

h

e

genome was derived when cells were exposed to low

temperatures

. Sequence analysis revealed a novel pathogenicity island termed

t

c

-PAI

Y

e

carrying insecticidal toxin genes that could be demonstrated to be transcriptionally silent

at body temperature, but to be essential for

Y

.

e

n

t

e

r

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c

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l

i

t

i

c

a

toxicity against insects at

low temperature. This data demonstrates a yet unknown

p

a

t

h

o

g

e

n

i

c

i

t

y phase of

Y.

enterocolitica

in insects, suggesting invertebrates as a potential

s

o

u

r

c

e

o

f pathogen

evolution.