ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND PHYSIOLOGICAL ...

241 Pages·2008·4.04 MB·English

by  Noha Mesbah

ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND
PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS
by
NOHA MOSTAFA MESBAH
(Under the Direction of Juergen Wiegel)
ABSTRACT
Halophilic alkalithermophiles are poly-extremophiles adapted to grow at high salt
concentrations, alkaline pH values and temperatures greater than 50ºC. Halophilic
alkalithermophiles are of interest from physiological perspectives as they combine unique
adaptive mechanisms and cellular features that enable them to grow under extreme conditions.
The alkaline, hypersaline lakes of the Wadi An Natrun, Egypt were chosen as sources for
isolation of novel halophilic alkalithermophiles. These lakes are characterized by saturating
concentrations of NaCl (5.6 M), alkaline pH (8.5-11) and temperatures of 50ºC due to intense
solar irradiation. The prokaryotic communities of three large lakes of the Wadi An Natrun were
assessed using 16S rRNA clone libraries. The Wadi An Natrun lakes are dominated by three
phylogenetic groups of Bacteria (Firmicutes, Bacteroidetes, α- and γ-proteobacteria) and two
groups of Archaea (Halobacteriales and Methanosarcinales). Extensive diversity exists within
each phylogenetic group; half of the clones analyzed did not have close cultured or uncultured
relatives. Three novel halophilic alkalithermophiles were isolated from the Wadi An Natrun. A
novel order, Natranaerobiales, was proposed to encompass these novel isolates. Natranaerobius
thermophilus was chosen as a model for more detailed physiological studies. Analysis of the
bioenergetic characteristics of N.thermophilus revealed the absence of cytoplasmic pH
homeostasis. Rather, N.thermophilus has the novel feature of maintaining the cytoplasmic pH at
a constant 1 unit below that of the extracellular pH, the cytoplasmic pH continuously changed
with the extracellular pH. To investigate the mechanism of this dynamic cytoplasmic pH
regulation, genes encoding putative cation/proton antiporter proteins were functionally
characterized. The physiological characteristics of these cation/proton antiporters were well
suited to the intracellular conditions of N.thermophilus. Collectively, antiporter proteins function
over the whole pH range for growth of N.thermophilus and display antiport activity over a wide
range of Na+ ion concentrations. Thus, they are capable of providing active cytoplasmic
acidification even if the intracellular environment of N.thermophilus changes. Most of the
antiporter proteins belong to the NhaC family of cation/proton antiporters; a group that is
primarily involved in expulsion of intracellular Na+. These results indicate that these antiporters
provide N.thermophilus tolerance to both alkaline pH and high salt concentrations.

INDEX WORDS: Halophile, Alkaliphile, Thermophile, Extremophile, Alkalithermophile,
Natranaerobiales, Natranaerobius thermophilus, Natronovirga, Wadi An
Natrun, Microbial diversity, Antiporter, Intracellular pH, Proton motive
force, Membrane potential, Phosphorylation potential
ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND
PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS

by

NOHA MOSTAFA MESBAH
B. Pharm. Sci., Suez Canal University, Egypt, 2002

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA
2008
© 2008
Noha Mostafa Mesbah
All Rights Reserved
ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND
PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS

by

NOHA MOSTAFA MESBAH

Major Professor: Juergen Wiegel

Committee: James T. Hollibaugh
Joy B. Doran Peterson
William B. Whitman

Electronic Version Approved:

Maureen Grasso
Dean of the Graduate School
The University of Georgia
December 2008
ACKNOWLEDGEMENTS
There are many people I would like to thank for helping make this dissertation possible,
either for helping me with my work, offering support, encouragement or both. I am thankful for
my advisor, Juergen Wiegel for his guidance and support. Juergen’s sustained encouragement
over the years has enabled me to reach my goals. I also appreciate the assistance of my
committee members, Tim Hollibaugh, Joy Peterson, and Barny Whitman. Many thanks to my
labmates, past and present: Boguslaw Lupa, Mohammed Salameh, Rob Onyenwoke, Yong Jin
Lee, Isaac Wagner, Elizabeth Burgess, and Karen Bowers, for keeping the energy level and
morale high. There are others at the University of Georgia who have helped me along the way
that I would like to thank – Lyla Lipscomb, James and Emily Henriksen, Magda Lupa, Dana
Cook, Susmitha Seshadri and Bijal Patel.
I would like to thank Dr. Greg Cook and all the members of the Cook lab at the Univesity
of Otago, New Zealand for helping me during my stay and for that very enjoyable cycling
weekend in Clyde.
Finally, I would like to thank my parents for encouraging me to pursue a career in science
and for their continued support of me as I have progressed forward in my life.

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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ........................................................................................................... iv
LIST OF TABLES ........................................................................................................................ vii
LIST OF FIGURES ....................................................................................................................... ix
CHAPTER
1 INTRODUCTION AND LITERATURE REVIEW .....................................................1
2 NOVEL AND UNEXPECTED PROKARYOTIC DIVERSITY IN WATER AND
SEDIMENTS OF THE ALKALINE, HYPERSALINE LAKES OF THE WADI
AN NATRUN, EGYPT ...........................................................................................51
3 NATRANAEROBIUS THERMOPHILUS GEN. NOV. SP. NOV., A HALOPHILIC,
ALKALITHERMOPHILIC BACTERIUM FROM SODA LAKES OF THE
WADI AN NATRUN, EGYPT, AND PROPOSAL OF NATRANAEROBIACEAE
FAM. NOV. AND NATRANAEROBIALES ORD. NOV. .....................................104
4 NATRONOVIRGA WADINATRUNENSIS GEN. NOV. SP. NOV. AND
NATRANAEROBIUS TRUEPERI SP. NOV., TWO HALOPHILIC,
ALKALITHERMOPHILIC MICROORGANISMS FROM SODA LAKES OF
THE WADI AN NATRUN, EGYPT ....................................................................126
5 BIOENERGETIC PROPERTIES AND INTRACELLULAR PH REGULATION IN
NATRANAEROBIUS THERMOPHILUS, AN ANAEROBIC, HALOPHILIC
ALKALITHERMOPHILIC BACTERIUM ..........................................................147
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6 ACTIVITY PROFILES OF NA+ (K+)/H+ ANTIPORTERS FROM THE
HALOPHILIC, ALKALITHERMOPHILIC NATRANAEROBIUS
THERMOPHILUS ARE ADAPTIVE TO THE EXTREME ENVIRONMENT ..167
7 CONCLUSIONS........................................................................................................200
APPENDICES .............................................................................................................................204
A CALCULATION OF BIOENERGETIC PARAMETERS .......................................204
B DEPENDENCE OF SOLUTE TRANSPORT ON NA+ IN NATRANAEROBIUS
THERMOPHILUS .................................................................................................210
C EFFECT OF PH ON PROTON PUMPING INTO INVERTED MEMBRANE
VESICLES ............................................................................................................212
D IN VIVO EXPRESSION OF NATRANAEROBIUS THERMOPHILUS
ANTIPORTER GENES ........................................................................................214
E EFFECT OF EXTRACELLULAR PH ON SOLUTE UPTAKE IN
NATRANAEROBIUS THERMOPHILUS ..............................................................222
F EFFECT OF EXTRACELLULAR PH ON INTRACELLULAR PH IN ENERGIZED
AND NON-ENERGIZED CELLS OF NATRANAEROBIUS THERMOPHILUS 225

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LIST OF TABLES
Page
Table 1.1: Definitions of different extremophiles ..........................................................................47
Table 2.1: Physiochemical properties of the water of the Wadi An Natrun lakes under study .....83
Table 2.2: Diversity and richness indices for bacterial clone libraries ..........................................84
Table 2.3: Distribution of bacterial OTUs into phylogenetic groups ............................................85
Table 2.4: Summary of OTUs affiliated with the α-proteobacteria ...............................................86
Table 2.5: Summary of OTUs affiliated with the γ- proteobacteria ..............................................87
Table 2.6: Summary of OTUs affiliated with the δ- proteobacteria, Bacteroidetes, Firmicutes,
and Spirochaetes ............................................................................................................88
Table 2.7: LIBSHUFF comparisons of bacterial clone libraries ...................................................89
Table 2.8: Diversity and richness indices for archaeal clone libraries ..........................................90
Table 2.9: LIBSHUFF comparisons of archaeal clone libraries ....................................................91
Table 3.1: Differential characteristics of strain JW/NM-WN-LFT and closely related species ..119
Table S3.1: PLFA composition of strain JW/NM-WN-LFT ........................................................122
Table 4.1: Selected characteristics that distinguish strains JW/NM-WN-LUT (and related strains)
and JW/NM-WN-LH1T from Natranaerobius thermophilus ......................................142
Table 4.2: Polar and neutral fatty acid composition of strains JW/NM-WN-LUT and JW/NM-
WN-LH1T ....................................................................................................................144
Table 6.1: Bacterial strains, plasmids and oligonucleotides ........................................................189
Table 6.2: List of predicted antiporter proteins from N. thermophilus ........................................192
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Table 6.3: Monovalent cation/proton antiport activity in inverted membrane vesicles of antiporter
expressing E.coli KNabc transformants ......................................................................194
Table 6.4: Apparent K values for N.thermophilus antiporter proteins .....................................195
0.5
Table 6.5: Cation/proton antiport activity of N. thermophilus antiporters as a function of pH ...196
Table D1: Oligonucleotides used in qRT-PCR, product sizes, optimal primer concentrations used
and PCR efficiency ......................................................................................................219
Table D2: Validation of reference genes .....................................................................................221

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