Adaptation of Acinetobacter Species
to Dry Environments
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim Fachbereich Bio Wissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main
von
Miriam Sand
aus München
Frankfurt, Mai 2016
(D30)
1. SUMMARY.................................................................................................................. 1
2. INTRODUCTION..........................
2.1 P r in c ip l e s
3
of o sm o a d a pta tio n in ba cteria ................................................................................................... 3
h x ON
2.1.1 Compatible solute-strategy .............................................. ........
2.1.2 Accumulation o f the compatible solute glycine betaine
2.1.3 Transport o f compatible solutes ............................................. .
2.2 T he genus A cinetobacter..................................................................................................................................... 12
2.3 A. BAYLYl AND A BAUMANNII - TWO REPRESENTATIVES OF THE GENUS ACINETOBACTER........................13
2.3.1 A. baumannii - a nosocomial pathogen ..... ..................................... ............. ....................................... 13
2.3.2 Multidrug resistances o f A. baumannii...................................................................................... „........ 15
2.3.3 Metabolic adaptation o f A. baumannii to hospital environments............ „............................... 19
2.3.4 Desiccation resistance o f A. baumannii........... ...........
21
2.3.5 Where does A. baumannii come from? ................................................................................................. 22
2.3.6 Acinetobacter baylyi - a nutritionally versatile soil bacterium .................................................24
2.3.7 Natural competence o f A. baylyi............................................................................................................. 25
2.3.8 Osmoadaptation o f A. baylyi....................................................................................................................... 28
2 .4. AIMS OF THIS THESIS.................................................................................................................................................. 2 9
3. ADDITIONAL RESULTS.......................................................................................30
3.1 A d a pta tio n o f A. ba um ann ii t o h o spit a l e n v ir o n m e n t s .......................................................................3 0
3.2 M a teria ls and m e t h o d s ........................................................................................................................................3 1
3.2.1 Bacterial strains and growth conditions............................................................................................... 31
3.2.2 Growth experiments ...........................................................................................................................
32
3.2.3 Quantification o f solutes......................... ......................................................................................... „........ 32
3.2.4 Desiccation assay........... „......................................................................... „....................................... „......„..33
3.2.5 2D-Electrophoresis.............................
34
3.2.5.1 Sample preparation.............................................................„.................................................................... ....34
3.2.5.2 Protein determination____________________________________________________________
34
3.2.5 3 First dimension: isoelectric focusing (IEF)________ ______________________ _______________ 35
3 2.5.4 Second dimension: SDS-PAGE_______________________________________ _____ _______ _______35
3.2.6 Peptide mass finger printing (PMF)..............................................
37
3.3 R e s u l t s .........................................................................................................................................................................3 9
3.3.1 Phosphatidylcholine serves as carbon source for A. baumannii.......................„........................3 9
3.3.2 The bet gene cluster is also present in A. baumannii_________________________ _________ 40
3.3.3 Glycine betaine is used as osmoprotectant in A. baumannii at high salinity ........................ 40
3.3.4 Desiccation resistance o f A. baumannii..........................................„....................„.................. ....... „..43
3 .3 .5 Proteins produced during desiccation stress in A. baumannii............................ ....................... 48
4. GENERAL DISCUSSION........................................................................................56
P ath w ays fo r m a n n ito l b io synth esis in n a t u re and m a n n ito l b io sy n th esis in A. baylyi
56
M a n n ito l as com pa tible solute a n d o sm o p r o t e c t a n t .......................................................................... 6 0
M a n n it o l as carbon a nd energy s o u r c e ....................................................................................................... 6 2
M a n n ito l as a radical sca v en g er .................................................................................................................... 6 3
ROLE OF A. baylyi M t l D in b io tech n o lo g y ..................................................................................................... 6 5
T he un iq u e bifu n ctio n a l MANNITOL-1-PHOSPHATE deh ydrogena se / ph o sph a ta se o f A. baylyi
67
4 .7 T he bet cluster in A cinetobacter- u pta k e o f ch o line a nd its conversion t o glycie b eta in e
( G B ).......................................................................................................................................................................................7 1
4.8 A d a pta tio n o f A. baumannii to d esic c a tio n .................................................................................................8 0
4.1
4.2
4.3
4 .4
4.5
4 .6
5. REFERENCES........................................................................................................... 87
8. PUBLICATIONS....................................................................................................109
8.1 "Mannitol, a compatible solute synthesized by A c in e to b a c te r b a y l y i in a two step pathway
INCLUDING A SALT-INDUCED AND SALT-DEPENDENT MANNITOL-1-PHOSPHATE DEHYDROGENASE"......... 1 0 9
8.2 "IDENTIFICATION AND CHARACTERIZATION OF AN OSMO-DEPENDENT AND OSMO-INDEPENDENT
CHOLINE TRANSPORTER IN ACINETOBACTER BAYLYI: IMPLICATIONS IN OSMOSTRESS PROTECTION AND
121
METABOLIC ADAPTATION"....................................
8.3 "MANNITOL-1-PHOSPHATE DEHYDROGENASES/PHOSPHATASES: A FAMILY of novel BIFUNCTIONAL
ENZYMES FOR BACTERIAL ADAPTATION TO OSMOSTRESS"..................................................................................... 1 3 5
9. DEUTSCHE ZUSAMMENFASSUNG..................................................................147
10. DANKSAGUNG....................................................................................................157
11. CURRICULUM VITAE........................................................................................159