Growth and Survival of
Acidithiobacilli in Acidic, Metal
Rich Environments
Stefanie Mangold
Department of Molecular Biology
Umeå 2012
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-474-4
Cover picture: 2D gel of Acidithiobacillus ferrooxidans, Stefanie Mangold
Electronic version available at http://umu.diva-portal.org/
Printed by: Department of Chemistry Printing Service, Umeå University
Umeå, Sweden 2012
In Memoriam Aviarum Mearum
Toni Klebingat, Barbara Mangold
Table of Contents
Table of Contents
Abstract
List of Abbreviations
List of Papers
Introduction
1.
Acidithiobacilli and Their Environments
1.1. Acidithiobacillus caldus and Acidithiobacillus ferrooxidans
1.2. Acidic Environments and Associated Microorganisms
2.
Applications and Implications
2.1. Biomining
2.2. Acid Mine Drainage
3.
Substrate Metabolism
3.1. Aerobic Iron Oxidation
3.2. Aerobic Sulfur Oxidation
3.3. Ferric Iron Reduction
4.
Metal Toxicity and Resistance
4.1. Metals and Toxicity
4.2. Metal Resistance Mechanisms
5.
Bioenergetics
5.1. Principles
5.2. Mechanisms for pH Homeostasis
6.
The Post-Genomic Era
6.1. Genetic Tools for Acidithiobacilli
6.2. ’Omics‘ Techniques and Continuous Culture
6.3. Systems Biology
Aims of This Study
Results and Discussion
Conclusions
Future Directions
Acknowledgements
List of References
i
ii
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3
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7
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53
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i
Abstract
Acidithiobacilli are acidophilic microorganisms that play important roles in
many natural processes such as acidification of the environment, influencing
metal mobility, and impacting on global sulfur and iron cycles. Due to their
distinct metabolic properties they can be applied in the industrial extraction
of valuable metals. Acidithiobacilli thrive in an environment which is
extremely acidic and usually low in organic carbon but highly polluted with
metals. In the quest to gain insight into how these microorganisms can thrive
in their extreme environment, relevant facets of metabolism, metal
resistance, and pH homeostasis were explored with the focus on two model
organisms, Acidithiobacillus caldus and Acidithiobacillus ferrooxidans.
Understanding these fundamental aspects of an acidophilic lifestyle will help
to eventually control detrimental effects on the environment due to
acidification and metal pollution as well as improving metal extraction
utilizing acidophilic microorganisms.
Bioinformatics can give information about the genetic capacity of an
organism. Likewise, ‘omics’ techniques, such as transcriptomics and
proteomics to study gene transcription profiles and differentially expressed
proteins can yield insights into general responses as well as giving clues
regarding specific mechanisms for adaptation to life in extreme
environments. This approach was used to investigate the sulfur metabolism
of At. caldus which is an important sulfur oxidizer for industrial metal
extraction. It was found that sulfur oxidation pathways were diverse within
acidithiobacilli and a model of At. caldus sulfur oxidation was proposed.
Furthermore, At. ferrooxidans anaerobic sulfur oxidation coupled to ferric
iron reduction was studied which can be of importance for industrial
processes. It was shown that anaerobic sulfur oxidation was, at least in part,
indirectly coupled to ferric iron reduction via sulfide generation. Moreover,
metal toxicity and resistance mechanisms in acidophiles are of major
interest. Thus, zinc toxicity in three model organisms, At. caldus,
Acidimicrobium ferrooxidans, and ‘Ferroplasma acidarmanus’, was
explored. An important finding was that the speciation of metals and other
chemical influences were of great importance for zinc toxicity in acidophiles.
Additionally, the three organisms showed distinct responses to elevated zinc
levels. Finally, the response of At. caldus to various suboptimal growth pH
was evaluated to gain insights into pH homeostasis mechanisms. The results
indicated that At. caldus used acid resistance mechanisms similar to those
described for neutrophilic microorganisms. Analysis of fatty acid profiles
demonstrated an active modulation of the cyctoplasmic membrane in
response to proton concentration, likely resulting in a more rigid membrane
at lower pH.
ii
List of Abbreviations
AMD
ARD
ars
ATP
BLM
CBB
ΔpH
ΔΨ
DMMM
F
FAME
GABA
GC
GSSH
Hdr
HMM
ICP-MS
IPB
LC
Maldi-ToF
MS
MW
NAD
NADH
NMR
NGS
nif
OMP
pHopt
pI
PMF
polyP
PTM
R
rus
Sat
sec pathway
semiQ RT-PCR
Acid mine drainage
Acid rock drainage
Operon encoding arsenic resistance
Adenosine triphosphate
Biotic Ligant Model
Calvin Benson Bassham cycle
pH gradient across a membrane
Membrane potential
Dynamic multi-species metabolic modeling
Faraday constant
Fatty acid methyl ester
γ-amino butyric acid
Gas chromatography
Sulfane sulfur
Heterodisulfide reductase
Hidden Markov Model
Indictively coupled plasma mass spectrometry
Iberian Pyrite Belt
Liquid chromatogaphy
Matrix assisted laser desorption ionization
Time of Flight
Mass spectrometry
Molecular weight
Nicotinamide adenine dinucleotide
Reduced NAD
Nuclear magnetic resonance
Next generation sequencing
Operon encoding nitrogen fixation
Outer membrane protein
Optimum growth pH
Isoelectric point
Proton motive force
Polyphosphate
Post translational modification
Gas constant
Operon encoding rusticyanine
ATP sulfurylase
Secretory pathway
Semi quantitative reverse transcriptase
polymerase chain reaction
iii
SDO
SMF
Sor
sox
SQR
STB
T
tat pathway
Tth/TetH
Topt
TQR
2D-PAGE
iv
Sulfur dioxygenase
Sodium motive force
Sulfur oxygenase reductase
Operon encoding sulfur oxidation genes
Sulfide quinone reductase
Stirred tank bioreactor
Absolute temperature
Twin arginine translocation pathway
Tetrathionate hydrolase
Optimum growth temperature
Thiosulfate quinone oxidoreductase
Two
dimensional
polyacrylamide
electrophoresis
gel
List of Papers
Paper I
Stefanie Mangold, Jorge Valdés, David S. Holmes and Mark Dopson (2011)
Sulfur metabolism in the extreme acidophile Acidithiobacillus caldus.
Frontiers of Microbiology: doi:10.3389/fmicb.2011.00017
Paper II
Héctor Osorio*, Stefanie Mangold*, Yann Denis, Ivan Nancucheo, D. Barrie
Johnson, Violaine Bonnefoy, Mark Dopson and David S. Holmes (submitted)
Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in the
extremophile Acidithiobacillus ferrooxidans.
*Authors contributed equally.
Paper III
Stefanie Mangold, Joanna Potrykus, Erik Björn, Lars Lövgren and Mark
Dopson (submitted) Extreme zinc tolerance in acidophilic microorganisms
from the bacterial and archael domains.
Paper IV
Stefanie Mangold, Venkateswara rao Jonna and Mark Dopson (manuscript)
Response of Acidithiobacillus caldus towards suboptimal pH conditions.
Paper not included in this thesis:
Carla Zammit, Stefanie Mangold, Venkateswara rao Jonna, Lesley Mutch,
Helen Watling, Mark Dopson and Elizabeth Watkin (2011) Bioleaching in
brackish waters – effect of chloride ions on the acidophile population and
proteomes of model species. Applied Microbiology Biotechnology 93: 319329.
v
vi
Introduction
In 2009, the Society for General Microbiology (SGM) awarded the Colworth
Prize Lecture to Prof. Geoffrey M. Gadd, University of Dundee, for his
contribution to geomicrobiology and understanding metal microbe
interactions (Gadd, 2010). This demonstrates the recognition and
appreciation of the importance that microbial actions have in geological
processes. In Gadd’s words, “The ubiquity and importance of microbes in
biosphere processes make geomicrobiology one of the most important
concepts within microbiology, and one requiring an interdisciplinary
approach to define environmental and applied significance and underpin
exploitation in biotechnology” (Gadd, 2010). Microorganisms interact with
metals and minerals in various ways and are involved in global cycling of all
elements. One group of microorganisms which especially impacts on the iron
and sulfur cycles as well as heavily influencing metal mobility in the
geosphere are acidophiles (optimum growth pH: pHopt < 5) and extreme
acidophiles (pHopt < 3). Previously, acidophiles mainly attracted interest due
to their use in industrial metal extraction, termed biomining. However, lately
they have also received attention due to their ability to live in an extreme
environment which might give clues to how extraterrestrial life could be like
(Parro et al., 2011). Acidophiles are also of high interest regarding the origin
of life on Earth as volcanic, low pH environments may have played an
important role for those processes (Holm and Andersson, 2005).
1. Acidithiobacilli and Their Environments
In this thesis, fundamental aspects of acidophilic lifestyle were studied by
investigating typical representatives of chemolithoautotrophic bacteria from
the Acidithiobacillacieae family. The acidithiobacilli inhabit extremely acidic
environments where they are part of a microbial community comprising
organisms from all domains of life. In the quest to gain some insight into the
question of how these microorganisms can thrive in their extreme
environment, relevant facets of metabolism, as well as metal resistance, and
pH homeostasis were explored with the focus on two model organisms,
Acidithiobacillus caldus and Acidithiobacillus ferrooxidans. In the following
sections the Acidithiobacillacieae family, especially At. caldus and At.
ferrooxidans, will be characterized in more detail, followed by a description
of typical acidic environments, and an overview of the acidophile microbial
community.
1
1.1. Acidithiobacillus caldus and Acidithiobacillus
ferrooxidans
In an effort to systemize their taxonomy, acidophilic species from the genus
Thiobacillus were renamed Acidithiobacillus (Kelly and Wood, 2000). The
genus Acidithiobacillus belongs to the class of γ-proteobacteria and today
comprises several species, i.e. At. albertensis (Bryant et al., 1983), At. caldus
(Hallberg and Lindström, 1994), At. ferrooxidans (Colmer et al., 1950), At.
thiooxidans (Waksman and Joffe, 1922), and the recently described coldtolerant At. ferrivorans (Hallberg et al., 2010). The acidithiobacilli are rodshaped Gram negative lithoautotrophs that fix carbon dioxide from the
atmosphere by the Calvin Benson Bassham cycle (CBB) (Elbehti et al., 2000;
Levican et al., 2008; Esparza et al., 2010). All acidithiobacilli can gain
energy from the oxidation of inorganic sulfur compounds but only At.
ferrooxidans and At. ferrivorans can utilize ferrous iron as well.
Additionally, other energy substrates such as molecular hydrogen (Drobner
1990) and formate (Pronk 1991) have been described.
Of all acidithiobacilli the mesophilic At. ferrooxidans (optimal growth
temperature, Topt = 30°C, and pHopt = 2.5) is the most studied species and it
is also one of the most studied acidophiles in general. The reason for this is
its early discovery and description (Colmer et al., 1950) and the fact that it
was thought to be the dominant species in many biomining processes and
natural environments (Brierley and Le Roux, 1978). More recent studies
have shown that At. ferrooxidans is not necessarily the dominant species but
rather, it is relatively easy to cultivate on solid medium which is why it was
selected for in many classical studies (reviewed in Johnson, 2001; Baker and
Banfield, 2003). As mentioned above, At. ferrooxidans can oxidize ferrous
iron as well as sulfur compounds and models of the respective metabolic
pathways have been established in some detail (Quatrini et al., 2009 and
references therein).
The first report of the moderate thermophilic At. caldus (Topt = 45°C,
pHopt = 2.5) was a description of its isolation from coal spoil heaps (Marsh
and Norris, 1983). Later it was characterized and announced as a new
species
(Hallberg and
Lindström, 1994).
At. caldus grows
lithoautotrophically with inorganic sulfur compounds as source of energy but
also mixotrophically with tetrathionate as electron donor along with glucose
and yeast extract (Hallberg and Lindström, 1994) as well as pyruvate (Aston
et al., 2011). Since its discovery At. caldus has been shown to efficiently
oxidize sulfur compounds during bioleaching (Dopson and Lindström,
1999), making it an important species involved in industrial bioleaching
using stirred tank bioreactors (STBs) at temperatures > 40ºC (reviewed in
Baker and Banfield, 2003; Rawlings, 2005). Okibe et al. (2003) showed that
At. caldus was the dominant sulfur oxidizer in early stages of bioleaching in
2
continuously operated STB and more recent studies on batch STB confirmed
these results (Dopson and Lindström, 2004; Hao et al., 2010; Zeng et al.,
2010). The important role in biomining spurred the interest to further study
At. caldus. Recently, the genome sequence of the type strain (ATCC 51756,
Valdes et al., 2009) and strain SM-1 (You et al., 2011) have been reported
which opened the door for a variety of investigations such as bioinformatic
and proteomic studies. Apart from its application in biomining, At. caldus
was also tested for possible application in remediation as a sorbent for
metals (Aston et al., 2010). Furthermore, its function as a biosensor for the
detection of toxic chemicals was demonstrated (Hassan et al., 2010).
1.2. Acidic Environments and Associated Microorganisms
Typical environments where acidophiles thrive are sulfate and metal rich,
acidic or extremely acidic sites worldwide. These sites can occur naturally or
be anthropogenic in origin. Acidic areas are often the result of the microbial
oxidation of sulfur or inorganic sulfur compounds and ferrous iron
eventually leading to the formation of sulfuric acid. Volcanic areas or
environments with high thermal activity are naturally occurring acidic sites
where elemental sulfur is formed by a condensation reaction of the
geothermal gases sulfur dioxide and hydrogen sulfide according to equation
(1).
SO2 + 2 H2S → 2 H2O + 3 S0
(1)
Whether microbial oxidation of sulfur results in acidification of the whole
site depends on the occurrence of basic minerals such as silicates or
carbonates which can neutralize the generated sulfuric acid. This also holds
true for environments which have been directly or indirectly influenced by
human activities to become acidic. Such places are typically rich in metal
sulfides which have been exposed to oxygen and water due to human mining
endeavors, e.g. abandoned mine sites or coal spoils. The most abundant
sulfide mineral is pyrite (FeS2) which can be efficiently dissolved to yield
sulfuric acid in a process catalyzed by microbes. Equation (2) describes the
overall reaction which simplifies the naturally occurring processes
(described in more detail in chapter 2.1).
4 FeS2 + 14 H2O + 15 O2 → 4 Fe(OH)3 + 8 H2SO4
(2)
A range of metal sulfides contain other metals than iron which are mobilized
by this dissolution process. At low pH, cationic metal ions are usually more
soluble than at higher pH values yielding the characteristic acid liquors with
a high content of dissolved metals. Another typical property of these
3
environments is their oligotrophic nature with a low content of soluble
organic carbon. Such anthropogenic acidic environments often found at
abandoned mining sites are termed acid mine drainage (AMD). One
extensively studied AMD site with an extremely low pH of below zero is Iron
Mountain in California, USA (Nordstrom and Alpers, 1999). Another well
studied man-made acidic environment is Rio Tinto in Spain where mining
activities date back to the ancient Romans. Besides the described
environments there are extremely acid, low-temperature and sulfur rich
environments such as sewers and sulfide enriched caves that are less well
studied (reviewed in Dopson and Johnson, 2012). Despite substantial
geochemical differences of sulfide rich caves and AMD environments, the
inherent microbial communities show remarkable resemblance (Jones et al.,
2012).
An unexpectedly large diversity of microorganisms from all domains of
life is able to populate these apparently hostile habitats. However, the
diversity in any particular acidic microenvironment is usually small with
only a few prokaryotic taxa comprising the microbial communities (reviewed
in Baker and Banfield, 2003; Schippers et al., 2010) and the same is true for
commercial biomining communities (reviewed in Rawlings, 2005; Schippers
et al., 2010). Most important for the microbial communities are sulfur and
iron oxidizing bacteria and archaea since they are responsible for the
prevailing acidity. However, acidophilic eukaryotes have also been detected
(reviewed in Baker and Banfield, 2003; Schippers et al., 2010) and their
roles and interactions with other community members are slowly being
unraveled (Amaral Zettler et al., 2002). Acidophilic flagellates, a ciliate, and
an amoeba were isolated from coal spoils and studied in vitro where they
were shown to predate acidophilic bacteria (Johnson and Rang, 1993).
Furthermore, a rather large eukaryotic diversity was found in Rio Tinto River
with photosynthetic algae contributing 60% of the biomass (Amaral Zettler
et al., 2002). Additionally, acidophilic fungi – including yeasts – were
isolated from AMD sites (Gadanho and Sampaio, 2006; Russo et al., 2008).
Often acidophilic bacteria and archaea are grouped according to their
optimal growth temperature into psychrotolerant (Topt < 15ºC), mesophilic
(Topt 15 - 40ºC), moderately thermophilic (Topt 40 - 60ºC), and extremely
thermophilic (Topt > 60ºC). Table 1 gives a short summary of selected
acidophiles together with their characteristics. Since acidic environments are
typically oligotrophic and often lack photosynthetic community members,
lithoautotrophic bacteria are frequently responsible for carbon fixation e.g.
the
acidithiobacilli.
But
also
heterotrophic
and
facultative
autothrophs/heterotrophs are usually part of acidophilic microbial
communities. Another element which might be scarce in acidic
environments and therefore, possibly growth limiting is nitrogen.
4
Table 1. Overview of selected acidophilesfrom all domains of life.
Organism
Acidianus ambivalens
Acidianus brierleyi
Acidimicrobium ferrooxidans
Acidiphilium cryptum
Acidithiobacillus albertensis
Acidithiobacillus caldus
Acidithiobacillus ferrivorans
Acidithiobacillus ferrooxidans
Acidithiobacillus thiooxidans
Cinetochilium spp.
Eutreptial spp.
Ferroplasma acidarmanus
Ferroplasma acidiphilum
Leptospirillum ferriphilum
Leptospirillum ferrodiazotrophum
Leptospirillum ferrooxidans
Metallosphaera prunae
Metallosphaera sedula
Picrophilus oshimae
Picrophilus torridus
Thermoplasma acidophilum
Sulfobacillus acidophilus
Sulfobacillus thermosulfidooxidans
Sulfolobus metallicus
Vahlkampfia spp.
a
Primary
carbon
source
Temperature
range
Energy
substratea
Domain/ Phylum
Reference
Heterotroph
Mixotroph
Mixotroph
Heterotroph
Autotroph
Autotroph
Autotroph
Autotroph
Autotroph
Heterotroph
Heterotroph
Heterotroph
Mixotroph
Autotroph
Autotroph
Autotroph
Mixotroph
Mixotroph
Heterotroph
Heterotroph
Heterotroph
Mixotroph
Mixotroph
Autotroph
Heterotroph
Thermophile
Thermophile
Mod. thermophile
Mesophile
Mesophile
Mod. thermophile
Psychrotolerant
Mesophile
Mesophile
Mesophile
Mesophile
Mesophile
Mesophile
Mesophile
Mesophile
Mesophile
Thermophile
Thermophile
Thermophile
Thermophile
Thermophile
Mod. thermophile
Mod. thermophile
Thermophile
Mesophile
S
S, Fe2+
Fe2+
Org. comp.
S
S
S
S, Fe2+
S
Org. comp.
Org. comp.
Fe2+
Fe2+
Fe2+
Fe2+
Fe2+
S
S
S
S
S
S, Fe2+
S, Fe2+
S
Org. comp.
Archaea/ Crenarchaeota
Archaea/ Crenarchaeota
Bacteria/ Actinobacteria
Bacteria/ α-Proteobacteria
Bacteria/ γ-Proteobacteria
Bacteria/ γ-Proteobacteria
Bacteria/ γ-Proteobacteria
Bacteria/ γ-Proteobacteria
Bacteria/ γ-Proteobacteria
Eucarya/ Ciliophora
Eucarya/ Euglenozoa
Archaea/ Euryarchaeota
Archaea/ Euryarchaeota
Bacteria/ Nitrospirales
Bacteria/ Nitrospirales
Bacteria/ Nitrospirales
Archaea/ Crenarchaeota
Archaea/ Crenarchaeota
Archaea/ Euryarchaeota
Archaea/ Euryarchaeota
Archaea/ Euryarchaeota
Bacteria/ Firmicutes
Bacteria/ Firmicutes
Archaea/ Crenarchaeota
Eucarya/ Percolozoa
Fuchs et al., 1996
Fuchs et al., 1996
Clark and Norris, 1996
Kusel et al., 1999
Bryant et al., 1983
Hallberg and Lindström, 1994
Hallberg et al., 2010
Colmer et al., 1950
Waksman and Joffe, 1922
Johnson and Rang, 1993
Johnson and Rang, 1993
Dopson et al., 2004
Golyshina et al., 2000
Coram and Rawlings, 2002
Tyson et al., 2005
Hippe, 2000
Fuchs et al., 1995
Huber et al., 1989
Schleper et al., 1995
Schleper et al., 1995
Segerer et al., 1988
Norris et al., 1996
Norris et al., 1996
Huber and Stetter, 1991
Johnson and Rang, 1993
S - inorganic sulfur compounds, Fe2+ - ferrous iron, Org. comp. - organic compounds.
This makes nitrogen fixation an important trait for acidophilic communities.
At. ferrooxidans as well as some members of the leptospirilli contain the
nitrogen fixing (nif) operon which encodes the enzyme nitrogenase
(NifHDG). Leptospirilli are mesophiles or moderate thermophiles belonging
to the order of Nitrospirales. They can only oxidize ferrous iron and have a
high affinity for this substrate which is one of the reasons why they often
outcompete At. ferrooxidans in commercial STB processes (reviewed in
Rawlings et al., 1999). Several species of leptospirilli have been isolated from
acidic environments: L. ferrooxidans (group I), L. ferriphilum (group II) and
L. ferrodiazotrophum (group III). It has been shown that L. ferriphilum is
not able to fix nitrogen whereas the opposite is true for the other species
(Coram and Rawlings, 2002; Tyson et al., 2005; Levican et al., 2008).
Another acidophilic iron oxidizer is the Gram positive Acidimicrobium
ferrooxidans which is moderately thermophilic and belongs to the
Actinobacteria. Likewise, sulfobacilli are Gram positive and moderately
thermophilic but they belong to the Firmicutes phylum and can only oxidize
inorganic sulfur compounds. Most of the above mentioned organisms are
autotrophs whereas the mesophilic Acidiphilium spp. belonging to the αproteobacteria are facultative heterotrophs. Those play an important role in
the microbial community as they can degrade organic compounds which
might be toxic to autotrophic members of the community. Furthermore,
archaea are often found in acidic environments especially at elevated
temperatures. Important members include several genera of the extremely
thermophilic, sulfur oxidizing family Sulfolobaceae, i.e. Sulfolobus,
Acidianus, Metallosphaera. Those belong to the Kingdom of Crenarchaeota
whereas the iron oxidizers Ferroplasma spp. and Picrophilus spp. belong to
the Thermophlasmatales in the Euryarchaeota kingdom. Some of the
archaea are heterotrophs, e.g. S. acidocaldarius and Ferroplasma spp..
2. Applications and Implications
The Rio Tinto mine in the Iberian Pyrite Belt (IPB) of southwest Spain is an
example of both the application of acidophiles for the benefit of humans as
well as detrimental environmental impact due to the same microbes. Already
the Romans extracted metals in this area without knowing that
microorganisms were involved in the solubilization of the metal ores.
Nowadays, Rio Tinto is known for its pollution due to AMD releasing acid
and high concentrations of metals into the environment. In both cases, the
same processes are responsible for the effects and in the following section
these processes as well as their applications and implications are described.
Understanding and ultimately manipulating the microorganisms involved in
those processes is key to improving applications and avoiding detrimental
effects and thus, an important aim of the research on acidophiles.
6
2.1. Biomining
The hunger of humans for metals dates back millennia to when we first used
metals to craft tools and ornaments. Since then the need for metals has
depleted the easily accessible high grade mineral ores and made it necessary
to explore ways to extract low grade and often refractive ores. Industrial
application of acidophilic microorganisms allows the extraction of low grade
ores when smelting is not cost efficient, is too environmentally costly, or
yields can be significantly increased by a microbial pre-treatment. This
process is generally called biomining. Strictly, biomining includes two more
specific processes, bioleaching and biooxidation, however, these terms are
often not used according to their specific definitions. Bioleaching comprises
all processes where acidophiles are utilized to catalyze the solubilization of
sulfide minerals and thereby release the metal of interest as soluble ions into
solution. A prominent example is copper extraction from the refractive metal
sulfide chalcopyrite (CuFeS2). In contrast, the term biooxidation describes
methods where the target metal is not part of the mineral but rather a
microbial pre-treatment makes it more accessible for extraction by other
methods. An example is gold extraction where biooxidation breaks down the
arsenopyrite (FeAsS) mineral lattice making gold particles more accessible
for extraction with cyanide, therefore yielding enhanced gold recovery.
The overall, simplified equation (2) for the dissolution of a typical metal
sulfide, pyrite (FeS2), is given in chapter 1.2. However, in the environment or
during biomining this is a complex multistep process which includes
reactions catalyzed by microorganisms as well as chemical reactions.
Schippers and Sand deciphered the mineral dissolution processes and
suggested two pathways for acid soluble (e.g. chalcopyrite) and acid
insoluble (e.g. pyrite) sulfide minerals (1999). The pathway for insoluble
metal sulfides was named according to the first sulfur intermediate,
thiosulfate, and is described with pyrite in equations (3) and (4). In this
pathway, sulfuric acid (sulfate (SO42-) and protons (H+)) is the main end
product.
FeS2 + 6 Fe3+ + 3 H2O → S2O32- + 7 Fe2+ + 6 H+
S2O32- + 8 Fe3+ + 5 H2O → 2 SO42- + 8 Fe2+ + 10 H+
(3)
(4)
Essentially, pyrite is dissolved by the chemical attack of ferric iron (Fe3+).
Iron oxidizing microorganisms catalyze this process by oxidizing the derived
ferrous iron (Fe2+) and thereby constantly regenerating the leaching
chemical as described in equation (5).
2 Fe2+ + 0.5 O2 + 2 H+ → 2 Fe3+ + H2O
(5)
7
In the case of acid soluble metal sulfides (MeS), the dissolution takes place
by ferric iron as well as protons and the main intermediates are polysulfides
and elemental sulfur. This pathway is called polysulfide pathway and is
illustrated in equations (6) and (7).
MeS + Fe3+ + H+ → M2+ + 0.5 H2Sn + Fe2+ (n ≥ 2)
0.5 H2Sn + Fe3+ → 0.125 S8 + Fe2+ + H+
(6)
(7)
In this pathway iron oxidizing microorganisms also regenerate the ferric iron
(5). But additionally sulfur oxidizers are important to oxidize the rather
stable sulfur intermediate as in equation (8) thereby also contributing to
acidification.
0.125 S8 + 1.5 O2 + H2O → SO42- + 2 H+
(8)
These processes have been applied to the biooxidation of gold (reviewed in
Rawlings et al., 2003) and uranium (Lange and Freyhoff, 1991; Lange et al.,
1991) as well as the bioleaching of copper (reviewed in Rawlings et al.,
2003), nickel (Riekkola-Vanhanen, 2010), and cobalt (Briggs and Millard,
1997). Although the underlying processes are identical the composition of
the metal sulfide is unique for each concentrate/ore and there are two
commonly utilized industrial set-ups for biomining, i.e. heap/dump leaching
and tank leaching. Consequently, the microbial communities responsible for
biomining vary in their composition according to the specific process
(reviewed in Johnson, 2001; Rawlings, 2005). Additionally, other industrial
processes have been employed, such as in situ leaching for uranium
(reviewed in Rawlings and Silver, 1995; Bosecker, 1997), but the two
processes mentioned above are those most commonly applied today.
Bioleaching heaps are comparably cheap and rather low technology
processes which make them an attractive method for the recovery of metals
from lower grade ores. Heap leaching has been mainly employed for the
recovery of copper (reviewed in Watling, 2006). The heaps can be anything
from simple waste dumps to sophisticatedly constructed heaps for improved
bioleaching. In any case the crushed ore is piled on an impervious base, and
supplied with an irrigation system for the acidic leach solution as well as a
collection system for the metal rich leachate. Aeration of the heap can be
passive or for improved oxygen supply the heap can be aerated by actively
blowing air into the base of the heap. Even with active aeration the oxygen
supply is commonly not homogeneous possibly leaving anaerobic zones
within the heap. Although bioleaching heaps do not need to be inoculated as
biomining microorganisms are inherently associated with the ore,
inoculation can shorten lag times and improve heap performance. Due to the
make-up of the heap leaching process microorganisms face gradients in pH,
8
temperature, and oxygen concentrations within the heap that are possibly
also influenced by seasonal changes. Thus, there are distinct ecological
niches within a bioleaching heap which will lead to a more diverse microbial
community compared to the more controlled tank leaching operations. The
latter are highly aerated, continuous flow stirred tank reactors which are
temperature and pH controlled. Usually a series of tanks are connected
where the first one is fed with the finely ground concentrate together with an
acidic phosphate and nitrogen enriched medium. Subsequently, the
suspension flows through the series of tanks within which the microbial
community catalyzes the dissolution of the metal sulfide. As the dissolution
proceeds in each tank of the series the conditions that the microbes face
change tank by tank leading to variations in the microbial community (Okibe
et al., 2003; Hao et al., 2010; Nancucheo and Johnson, 2010). Tank leaching
processes are most commonly applied for the biooxidation of gold but have
also been used for the bioleaching of cobalt and the applicability for
bioleaching of nickel has been demonstrated (reviewed in Norris et al.,
2000; Rawlings et al., 2003).
2.2. Acid Mine Drainage
The processes described in the former section can give rise to detrimental
environmental effects after mining sites are abandoned leading to AMD or
when metal sulfide rich deposits are naturally exposed to oxygen and water
leading to acid rock drainage (ARD). In the following, the term AMD will be
used but the same concepts generally apply to ARD. Similar to bioleaching
liquids, AMD liquors are rich in sulfate, ferric iron, and other metals as well
as being acidic, sometimes extremely so. AMD liquors are often discharged
into the environment without control. This can lead to acidified and metal
polluted groundwater, rivers, streams, and sediments, threatening aquatic
life as well as vegatation. The mining industry has recognized AMD as a
serious problem related to their activities and efforts for risk assessment and
AMD control are made (Akcil and Koldas, 2006). The generation of AMD as
well as its dimensions are not only dependent on the chemical and microbial
processes mentioned above, but are influenced by many factors including
mineralogy, hydrology, geology, and geochemistry making risk assessment
and AMD predictions a complex and difficult task (reviewed in Nordstrom,
2011). Especially important for the risk assessment of AMD is the
consideration of metal toxicity for the surrounding biota as large amounts of
metals can be mobilized. For example, the rivers Rio Tinto and Odiel
impacted by AMD from the IPB transport about 37% and 15% of global gross
flux (from rivers to the ocean) of dissolved zinc and copper, respectively
(Nieto et al., 2007). To aid this risk assessment the mobility of metals at
AMD sites can be predicted taking into account metal chemical, physical,
9
and geochemical information (Smith, 2007, see also chapter 4.1). The
contamination in the affected areas constituting high toxicity for the biota
has been described (Schippers et al., 2000; Nieto et al., 2007).
Due to its damaging effects several strategies for control and remediation
of AMD have been developed (reviewed in Johnson and Hallberg, 2005). In
principle, the strategies can be divided into source- and migration control
where the former is preferred but often is not feasible. An attempt to control
AMD generation at the source is, for example, the application of biocides to
inactivate the AMD generating microorganisms (for a review on the
microbial communities in AMD see Baker and Banfield, 2003; Schippers et
al., 2010), however this proved not to be very effective (Loos et al., 1989;
Sand et al., 2007). If it is not possible to avoid the generation of AMD at the
source there are a range of strategies to control and remediate it such as
chemical treatments to neutralize the AMD liquors (e.g. addition of lime), or
biological treatments (e.g. wetlands) (Johnson and Hallberg, 2005).
3. Substrate Metabolism
As mentioned in chapter 1, the main energy substrates utilized by
acidithiobacilli are inorganic sulfur compounds and ferrous iron. Some
details about the metabolic pathways of those substrates are known,
especially for At. ferrooxidans. The following sections give a summary of
aerobic iron and sulfur oxidation as well as anaerobic sulfur oxidation
coupled to ferric iron reduction in acidithiobacilli.
3.1. Aerobic Iron Oxidation
Iron in the environment is mostly in two oxidation states, ferrous (Fe2+) and
ferric iron (Fe3+). The role of prokaryotes in the iron cycle including ferrous
oxidation and ferric reduction is reviewed in Bird et al. (2011). The dominant
form of iron in the environment depends on chemical parameters such as
oxygen concentration, pH, and redox potential. Ferrous iron is stable in
anoxic conditions but rapidly oxidized to ferric iron at neutral pH in the
presence of molecular oxygen. On the other hand, ferrous iron oxidation
rates are very low at acidic pH. Furthermore, the redox potential of the
ferrous/ferric couple depends on the solubility of the ions and therefore, it
strongly depends on pH and the presence of chelating agents. The solubility
of ferric iron is particularly influenced by pH as it is only completely soluble
at pH < 1.6 or in presence of chelating agents such as citrate. Yet it is the
redox potential that fundamentally impacts on the utilization of iron as an
energy substrate. At acidic pH with soluble ferrous and ferric iron the redox
potential of the couple is + 780 mV. At neutral pH the redox potential of the
soluble, ligand bound species is much more negative making iron a more
10
favorable substrate at neutral pH. However, at the same time, the redox
potential of the oxygen/water couple is also more positive at low compared
to neutral pH, i.e. oxygen becomes a more favorable electron acceptor at
acidic pH. All in all aerobic iron oxidation at low pH can only harness a
potential of around 350 mV and can thus yield a small amount of energy.
Despite this At. ferrooxidans, At. ferrivorans, and many other acidophiles
from various phyla are iron oxidizers, possibly due to the high abundance of
iron in their natural habitat (for a general review on iron oxidizing
prokaryotes see Hedrich et al., 2011; while acidophiles are reviewed in
Bonnefoy and Holmes, 2012).
Figure 1. Aerobic iron oxidation in At. ferrooxidans. Electron transport components involved in the
‘downhill’ pathway from ferrous iron to oxygen are: (i) Cyc2, a cytochrome c located at the outer membrane;
(ii) a periplasmic blue copper protein rusticyanin A (RusA); (iii) Cyc1, a cytochrome attached to the
periplasmic side of the cytoplasmic membrane; and (iv) a type aa3 terminal cytochrome oxidase. The ‘uphill’
pathway components include: (i) a cytochrome bc1 complex (encoded within operon petI); (ii) the quinone
pool; and (iii) the NADH1 dehydrogenase complex. Dotted lines indicate the electron pathway and arrows
show the proton fluxes.
A model for At. ferrooxidans iron oxidation has been suggested (Ingledew,
1982), studied (Appia-Ayme et al., 1999), and recently refined (Quatrini et
al., 2009) and is broadly accepted today (Figure 1). Yet, lately it has been
realized that this model cannot serve as a universal acidophile iron oxidation
strategy since more than one pathway has evolved in this environment
(Amouric et al., 2011; Bonnefoy and Holmes, 2012). The most striking
difference between At. ferrooxidans and the newly announced species At.
ferrivorans is the psychrotolerance of the latter. Additionally, both species
utilize different iron oxidation pathways (Hallberg et al., 2010; Amouric et
11
al., 2011; Bonnefoy and Holmes, 2012), a finding that was confirmed by
deciphering the At. ferrivorans genome sequence (Liljeqvist et al., 2011).
This led to the suggestion that the iron oxidizing acidithiobacilli actually
comprise four species instead of two (Amouric et al., 2011). Nevertheless, all
species face the challenge of the positive redox potential of the ferrous/ferric
couple (780 mV) posing particular implications for the acidithiobacilli which
fix carbon by the CBB cycle. This pathway requires the reducing power from
NADH but the redox potential of NAD+/NADH (-320 mV) is much more
negative compared to that of the ferrous/ferric couple at low pH (780 mV).
To overcome this discrepancy acidithiobacilli partition the electron flow and
drive an ‘uphill’ pathway to gain the reducing power of NADH plus a
‘downhill’ pathway for gaining energy in form of adenosine triphosphate
(ATP) by transferring electrons to the final acceptor molecular oxygen
(Figure 1). In At. ferrooxidans, the electron transport components involved
in the ‘downhill’ pathway from ferrous iron to oxygen are: (i) Cyc2, a
cytochrome c located at the outer membrane; (ii) a periplasmic blue copper
protein rusticyanin A (RusA); (iii) Cyc1, a cytochrome c4 attached to the
periplasmic side of the cytoplasmic membrane; and (iv) a type aa3 terminal
cytochrome oxidase. It is believed that the splitting of the electron flow into
‘uphill’ and ‘downhill’ pathways takes place at the rusticyanin. The ‘uphill’
pathway components include: (i) a cytochrome bc1 complex (encoded within
operon petI); (ii) the quinone pool; and (iii) the NADH1 dehydrogenase
complex. In contrast, At. ferrivorans contains a high potential iron sulfur
protein, Iro, which is suggested to be the iron oxidase (Amouric et al., 2011)
as well as two rusticyanin isoforms, rusA and rusB.
3.2. Aerobic Sulfur Oxidation
Sulfur occurs naturally in eight oxidation states. Elemental sulfur has an
oxidation state of zero but sulfur compounds range from -2 (sulfide) to +6
(sulfate and sulfuric acid). All acidithiobacilli can oxidize elemental sulfur
and inorganic sulfur compounds such as sulfide (S2-), thiosulfate (S2O32-),
tetrathionate (S4O62-), or sulfite (SO32-). In contrast to the oxidation of
ferrous iron, sulfur oxidation yields much more energy (Pronk et al., 1990).
It was realized in the 1980’s that the sulfur oxidation pathways of acidophiles
and even within the acidithiobacilli are not the same (Kelly, 1985) which was
also highlighted by investigating the genome sequences of At. ferrooxidans,
At. caldus, and At. thiooxidans (Valdes et al., 2008a). The sulfur oxidation
model suggested for At. ferrooxidans (Quatrini et al., 2009) includes a range
of enzymes and cytochrome complexes dealing with the various sulfur
compounds and delivering electrons to molecular oxygen as the terminal
electron acceptor. The periplasmic side of the outer membrane contains
tetrathionate hydrolase (Tth or TetH, both designations can be found in
12
literature) which hydrolyzes tetrathionate to thiosulfate, sulfur, and sulfate.
Thiosulfate is converted to tetrathionate by thiosulfate quinone
oxidoreductase (TQR) which is located in the cytoplasmic membrane. At the
same location is sulfide quinone reductase (SQR) that oxidizes sulfide to
yield polysulfides or sulfur while the cytoplasm contains heterodisulfide
reductase (Hdr) and ATP sulfurylase (Sat). The enzyme complex HdrABC
has been studied in methanogenic archaea where it reversibly reduces the
disulfide bond in heterodisulfide (Hedderich et al., 2005). However, in At.
ferrooxidans this enzyme complex is proposed to oxidize the disulfide bond
of sulfane sulfur (GSSH) derived from elemental sulfur since several
characteristics support its role in sulfur oxidation (discussed in Quatrini et
al., 2009). How sulfite, produced by HdrABC, is further oxidized to the end
product sulfate is not completely clear but Sat is hypothesized to be involved
(Quatrini et al., 2009). Electrons derived from GSSH, thiosulfate, or sulfide
enter the quinone pool and are subsequently transferred to either the
NADH1 complex to yield reducing power or to the terminal oxidases. There
are several cytochromes and cytochrome complexes predicted to be involved
in the electron transport chain (Quatrini et al., 2009). The terminal oxidases
bd or bo3 probably receive electrons directly from the quinone pool whereas
the aa3 oxidase possibly receives them indirectly through the bc1 complex
(encoded in the petII operon) and a cytochrome c or a high potential ironsulfur protein. It is interesting to note that there are two differentially
expressed bc1 complexes in the At. ferrooxidans electron transport chain
(Appia-Ayme et al., 1999; Brasseur et al., 2002; Bruscella et al., 2007). The
bc1 complex encoded within the petI operon is expressed during growth on
ferrous iron whereas the second complex encoded in the petII operon is upregulated during sulfur oxidation. Further, it has been shown that the former
is involved in the ‘uphill’ pathway towards the NADH1 complex and the
latter in the ‘downhill’ pathway towards the terminal oxidase. Although the
current model of sulfur oxidation in At. ferrooxidans is able to explain many
experimental observations it is still not understood how the insoluble,
hydrophobic elemental sulfur is made accessible for the cell. The current
hypothesis is that it is converted to GSSH and then oxidized by Hdr in the
cytoplasm, but how this is accomplished in detail remains unclear.
Sulfur oxidation in At. caldus has been previously studied (Hallberg et al.,
1996; Dopson et al., 2002; Bugaytsova and Lindström, 2004; Rzhepishevska
et al., 2007). In the early studies, the sulfur compounds oxidized and the
cellular compartment in which the reaction takes place were revealed
(Hallberg et al., 1996). Furthermore, it was demonstrated that ATP
production is solely due to electron transport phosphorylation and not
substrate-level phosphorylation (Dopson et al., 2002). Those studies yielded
a preliminary model showing the oxidation of thiosulfate, tetrathionate,
sulfur, sulfide, and sulfite in a cellular context without relating the reactions
13
to specific enzymes (Hallberg et al., 1996). The subsequent two studies
(Bugaytsova and Lindström, 2004; Rzhepishevska et al., 2007) concentrated
on Tth and its regulation. Tth was purified and characterized; furthermore, it
was shown to be a soluble, periplasmic homodimer (Bugaytsova and
Lindström, 2004). The Tth gene cluster is suggested to be transcriptionally
regulated (Rzhepishevska et al., 2007). Additionally, a comparison of the At.
caldus draft genome sequence with the genome sequence of At. ferrooxidans
showed that the former harbors sulfur oxidation enzymes which are not
present in At. ferrooxidans (Valdes et al., 2008a). This suggests that both
species use quite distinct pathways. A detailed model of At. caldus sulfur is
lacking and therefore, requires further investigation. In the meantime, the
availability of the At. caldus’ genome sequence favored a global proteomic
and bioinformatic approach to study its sulfur metabolism. Consequently,
one of the aims of this thesis was to further investigate this pathway and
suggest a more detailed model including proposed enzymes. This model will
be presented in paper I. Very recently, after publication of paper I, a more
refined sulfur oxidation model for At. caldus MTH-04 has been published
(Chen et al., 2012). This new model is discussed in more detail in the results
section for paper I.
3.3. Ferric Iron Reduction
In contrast to ferrous iron oxidation, the utilization of ferric iron as a
terminal electron acceptor by acidophiles, i.e. ferric reduction, has not been
well studied. Yet, this reaction is of great importance in the environment as
well as for biotechnological applications (reviewed in Johnson, 1998). In
AMD environments as well as in heap or stirred tank bioleaching operations
soluble ferric iron occurs in high concentrations. At the same time
microaerophilic or anaerobic zones might be present especially in
abandoned mines or bioleaching heaps (Johnson et al., 1993). As discussed
above, the redox potential of the ferrous/ferric couple is 780 mV at pH ~ 2
rendering ferric iron a favorable electron acceptor at acidic conditions. The
reduction of ferric iron can be coupled with the oxidation of various energy
substrates such as organic compounds, elemental sulfur, inorganic sulfur
compounds, and hydrogen (Das et al., 1992; Pronk et al., 1992; Bridge and
Johnson, 1998; Ohmura et al., 2002).
Respiration utilizing ferric iron coupled to the oxidation of organic
compounds is widespread in pH neutral conditions and has been well
studied especially in anaerobic Geobacter spp. and Shewanella spp.
(reviewed in Lovley, 2008; Bird et al., 2011). In acidic environments a range
of microorganisms have been reported to be capable of utilizing ferric iron as
well. However, those acidophilic microorganisms are mostly facultative
anaerobes rather than obligate anaerobes and some grow poorly in
14
completely anoxic conditions (Kusel et al., 1999; Coupland and Johnson,
2008). The first mention of ferric reduction by acidophiles was in 1976
(Brock and Gustafson) although it remained unclear whether the tested
organisms, At. ferrooxidans, At. thiooxidans, and Sulfolobus acidocaldarius
were able to grow anaerobically. That anaerobic sulfur oxidation with ferric
iron as electron acceptor is able to yield enough energy to support growth
was subsequently demonstrated for At. ferrooxidans (Das et al., 1992; Pronk
et al., 1992). Ohmura et al. (2002) showed that both ferric and sulfur
reduction could be coupled to hydrogen oxidation as primary energy source
for At. ferrooxidans under anaerobic conditions. In contrast, Gram positive,
moderately thermophilic bacteria such as Sulfobacillus spp. and
Acidimicrobium ferrooxidans performed best when grown heterotrophically
or mixotrophically although some species were able to utilize tetrathionate
as electron donor as well (Bridge and Johnson, 1998). The ability to couple
the oxidation of organic compounds to ferric iron reduction has been
reported for the acidophilic heterotroph Acidiphilum cryptum (Kusel et al.,
1999) and has recently been studied in several other acidophilic heterotrophs
such as Acidocella, Acidobacterium-like, Acidispheara, and Frateuria-like
spp. (Coupland and Johnson, 2008). The latter study suggested that this
trait is widespread in acidophilic heterotrophic bacteria. Additionally,
species from the archaeal genus Ferroplasma can respire ferric iron with
yeast extract as electron donor (Dopson et al., 2007). Although it is now
recognized that facultative anaerobic growth by dissimilatory ferric
reduction is an important metabolic capacity of several acidophiles, the
molecular details remain enigmatic. Attempts to elucidate the anaerobic
electron transport chain in acidophiles suggest that cytochromes c and b are
involved in Ferroplasma spp. (Dopson et al., 2007). Additionally, a new
cytochrome was isolated from anaerobically grown At. ferrooxidans which
showed specific characteristics supporting its role in the anaerobic electron
transport chain (Ohmura et al., 2002). Recently, ferric reduction activity was
associated with Tth from At. ferrooxidans (Sugio et al., 2009) however, this
finding has been questioned. Additionally, ferric reductase activity of the At.
ferrooxidans ArsH protein was reported (Mo et al., 2011). Althought the
authors conceded that the biochemical function of ArsH was not
unequivocally elucidated they proposed ArsH to be a cytoplasmic ferric iron
reductase (Mo et al., 2011). Another recent study applying a proteomics
approach to compare aerobic and anaerobic sulfur oxidation of At.
ferrooxidans suggested that rusticyanin and a cytochrome c also encoded in
the rus operon are involved in the anaerobic electron transport chain
(Kucera et al., 2012). However, until now convincing evidence for the
identity of an acidophilic ferric reductase is missing. Therefore, we
attempted to expand the knowledge of anaerobic ferric reduction by At.
ferrooxidans in a study presented with paper II.
15
Table 2. Metal concentrations of selected metals measured at three different AMD sites. The data exemplifies variations in
metal concentrations depending on the AMD site, the geochemical conditions as well as seasonal changes.
Location
Richmond mine, Iron Mountain, USAa
Richmond mine, Iron Mountain, USAa
Richmond mine, Iron Mountain, USAa
Tinto river, Spain; Minimum valuesb
Tinto river, Spain; Maximum valuesb
Odiel river, Spain; Minimum valuesb
Odiel river, Spain; Maximum valuesb
Bozinta tailings heap, Romaniac
Bozinta tailings heap, Romaniac
Bozinta tailings heap, Romaniac
a Measurement
Fe (total)
[mg/L]
20300
86200
111000
0.07
2804
0.31
22.5
0.35 (0.36)
1108 (1229)
<0.1 (0.07)
Cu
[mg/L]
290
2340
4760
0.2
84.3
0.5
17.1
1 (3)
25 (47)
0.33 (0.6)
Ni
[mg/L]
0.66
2.9
3.7
16
742
19
500
n.d.d
n.d. d
n.d. d
Co
[mg/L]
1.3
15.5
5.3
52
3754
33
938
n.d. d
n.d. d
n.d. d
Zn [mg/L]
2010
7650
23500
2.2
152.3
1.3
36.4
9 (27)
116 (112)
0.21 (0.33)
As (total)
[mg/L]
56.4
154
340
<3
2290
<3
22
38 (42)
58 (52)
3.4 (8.2)
Sulfate
[mg/L]
118000
360000
760000
150
5547
110
2379
n.d. d
n.d. d
n.d. d
pH
0.48
-0.7
-2.5
2.22
5.01
2.95
5.05
7.0 (1.3)
3.7 (1.3)
8.3 (0.3)
of three extremely acidic samples taken during 1990-1991 from the underground Richmond mine (Nordstrom and Alpers,
1999).
b Minimum and maximum values from weekly measurements over a period from February 2002 until September 2004 (Nieto et al., 2007).
c Mean values (standard deviation) from several samples of three different sites sampled in October 1996, May 1997, October 1997 and July
1998 (Schippers et al., 2000).
d n.d. not determined.
4. Metal Toxicity and Resistance
In section 2.2 it was already touched upon that mobility of metals in the
environment depends on many different factors (Smith, 2007; Nordstrom,
2011). Therefore, it is easily conceivable that the concentrations of metals
differ largely between various AMD sites and biomining processes.
Nevertheless, concentrations of soluble metal ions are typically high in acidic
environments, often in the range of mg/L up to g/L (Table 2). Confronted
with such high amounts of metals it seems surprising that microorganisms
can thrive in those environments. In fact, some acidophiles, especially
archaeal species, are not very tolerant to some metals and thus their use in
biomining operations is hampered (Dopson et al., 2003). In the following
chapter some theoretical considerations on metals and metal toxicity as well
as a short overview of studies on acidophile metal resistance are given.
4.1. Metals and Toxicity
Chemically metals are defined as “elements which conduct electricity, have a
metallic luster, are malleable and ductile, form cations, and have basic
oxides” (Atkins and Jones, 1997). This definition includes a large range of
elements and further categorization is needed in order to highlight specific
properties of metals. Such grouping should be based on chemical or physical
qualities to ensure a sound and unambiguous terminology (Duffus, 2002). If
used alone, terms like essential metal, trace metal, or toxic metal lack
precision since it depends on the organism and the environment whether a
metal is essential, available in trace amounts, or toxic. For example, metals
such as iron, copper, and zinc are essential for most microorganisms as they
are important for the function of many enzymes. Nonetheless, even those
metals can exert a toxic effect if they exceed a certain concentration which
again depends on the organism. The toxicity of a metal primarily depends on
its interaction with the organism, i.e. interactions or reactions with
biomolecules. Thus, the chemical and physical properties of the metal can
help to predict its toxicity (Newman et al., 1998; Ownby and Newman,
2003). Chemical and physical properties are for example: oxidation state
(represents the charge of the element when electrons are taken into
account), ionic radius (size of the element) or electronegativity (strength of
an element to attract electrons). Based on those properties metals can be
categorized and a useful categorization referring to the lewis acid character
and preferred ligands has been developed (Ahrland et al., 1958) and later
refined (Pearson, 1963, 1968b, a).
17
Table 3. Characteristics and classifications of selected metals.
Element
(chemical symbol)
Iron (Fe)
Copper (Cu)
Zinc (Zn)
Nickel (Ni)
Cobalt (Co)
Arsenic (As)
Cadmium (Cd)
Silver (Ag)
Lead (Pb)
Mercury (Hg)
Gold (Au)
a Electronegativity
Periodic
table
d block
Class A/Bb
Mobilityc
borderline (Fe2+),
A (Fe3+)
B (Cu+),
borderline (Cu2+)
borderline
++
+++
d block
Electronegativitya
1.7 (Fe2+),
1.8 (Fe3+)
1.8 (Cu+),
2.0 (Cu2+)
1.65
d block
1.91
borderline
+++
d block
1.88
borderline
+++
p block
borderline (As3+)
++
d block
d block
2.0(As3+),
2.2 (As5+)
1.69
1.93
B
B
+++
+
p block
1.6 (Pb2+)
d block
1.8 (Hg+),
2.0 (Hg2+)
2.54 (Au+),
2.9 (Au3+)
B (Pb2+),
borderline (Pb4+)
B
B
d block
d block
+++
Recovered by
biomining
Not commercially
Bioleaching
(mostly heaps)
Bioleaching
(few operations)
Bioleaching
(few operations)
Bioleaching
(few operations)
Not commercially
Biological significance
Important trace metal for
many organisms
Often required in trace
amounts
Usually required in trace
amounts
Can be utilized in
biological systems
Can be utilized in
biological systems
Often toxic
Often toxic
Often toxic
+
Not commercially
Biooxidation
(tank leaching)
Not commercially
++
Not commercially
Often toxic
n.a.d
Biooxidation
(tank leaching)
Usually no significance
Often toxic
according to Pauling scale (Pauling, 1960; data from Smith, 2007).
according to Lewis acidity in class A (hard), class B (soft), and borderline metals (data from Duffus, 2002).
c Relative mobility under oxidizing conditions pH < 3 (data from Smith, 2007). Ranking: + somewhat mobile, ++ mobile, +++ very mobile.
d n.a. not available.
b Classification
A lewis acid is an element which can act as electron acceptor and metals are
grouped in three classes according to their preferred ligand: class A or ‘hard
acids’, class B or ‘soft acids’, and borderline. Class A (hard) ions have a hard,
non-polarizable electronshell and prefer alike hard ligands, such as oxygen
donors, with which they assume mostly ionic bonding where they are easily
displaced. In contrast, class B (soft) ions are more polarizable and prefer soft
ligands, such as sulfur or sulfide donors, with which they form more covalent
bonding and are thus less mobile. Since class B metals prefer ‘soft’ ligands
such as sulfur or sulfide donors they can interfere largely with cellular
components causing toxic effects. However, when using those categories it
needs to be keept in mind that the classes apply to a specific ionic form and
the borderline metals are difficult to classify and thus the classification is not
absolute. Likewise, the knowledge of metal properties can be used to make
qualitative statements about relative metal mobilities for a certain
geochemical condition (Smith, 2007). Table 3 summarizes some metal
characteristics and categorizations as well as giving information whether the
metal is recovered by biomining.
Figure 2. Metal toxicity in microorganisms depends on (i) bioavailability; (ii) interactions with ions at the
binding site; (iii) transport into the cell; and (vi) the reactivity of the metal with biomolecules. Usually the free
metal cation is the most toxic form of the metal.
It is not only the reactivity with biomolecules that determines the toxicity of
a metal. Before the metal can exhibit its effects it needs to enter the cell.
Therefore, several other aspects need to be considered (Figure 2) including
(i) bioavailability of the metal; (ii) interactions with other ions at the binding
site; and (iii) transport into the cell. Bioavailability denotes the accessibility
of a substance for organisms, e.g. a solid substance is often unusable for
microorganisms. Thus, bioavailability of metals correlates with metal
speciation which itself depends on the characteristics of the metal and
solution chemistry. Generally, the free metal ion is believed to be the most
toxic species as complexed metals are often not bioavailable. Assuming that
19
the free metal cation is the only toxic species it still needs to be considered
that this cation can interact with other cations at the binding or up-take site.
Other cations in solution, such as Ca2+ or H+, might compete with the free
metal ion and thus mitigate its toxicity. However, care needs to be taken as
the assumption that the free metal cation is the only toxic species is not true
in all cases. For example it has been shown that the complexed zinc in
ZnHPO40(aq) could be taken up by Arthrobacter and therefore, this species
significantly contributed to zinc toxicity (Moberly et al., 2010). These
considerations are incorporated in the so-called Biotic Ligand Model (BLM)
used for modeling metal toxicity in aquatic organisms (reviewed in Di Toro
et al., 2001). In the BLM the biotic ligand replaces the site of toxic action in
the aquatic organism, e.g. gills in the case of fish. In the case of
microorganisms the toxic metal has to be transported into the cell to show its
toxic effect. Thus, the transport of the toxic species over the cell membrane is
of great importance requiring special attention. Often unspecific,
constitutively expressed importers are the cause of toxicity as influx of the
toxic ion through those transporters cannot be stopped (Nies, 1999).
Taking into account all aspects for single metal toxicity already gives a
quite complex picture; however, often it is necessary to consider metal
mixtures. Especially in the case of AMD or biomining sites multiple metals
are the rule and not the exception. Attempts have been made to model the
toxicity of metal mixtures and illustrate the complexity of the phenomenon
(Ownby and Newman, 2003; Kamo and Nagai, 2008).
4.2. Metal Resistance Mechanisms
Microorganisms have evolved mechanisms to counteract metal toxicity,
firstly by establishing metal homeostasis and where this is insufficient by
resistance mechanisms. Unlike toxic organic compounds, metals cannot be
degraded to detoxify them. Consequently, there are limited strategies for
metal resistance (Nies, 1999, 2003): (i) efflux of the metal out of the cell; (ii)
sequestration; (iii) blocking the permeation/up-take into the cell; and (iv)
reduction to a less toxic ionic form. As discussed for metal resistance in
neutrophiles such as the extremely metal resistant Cupriavidus
metallidurans the most important strategy is the transport of excess metals
out of the cell (reviewed in Nies, 1999, 2003; von Rozycki and Nies, 2009).
Metal sequestration is often unfavorable as it is energetically expensive to
either import or synthesize the sequestration ligands. Blocking of metal ions
from entering the microbial cell can be achieved by alterations in the cellular
membrane to prevent leakage of metal ions into the cell as reported for At.
ferooxidans in response to copper and nickel (Mykytczuk et al., 2011). The
reduction of metals to a less toxic form is an elegant way for detoxification
but only few metal ions can be reduced within the cell due to their favourable
20
redox potential. Merely in the case of mercury reduction suffices for
detoxification since Hg2+ is reduced to the volatile Hg0 which can leave the
cell by diffusion. This reduction is carried out by mercury reductase MerA
encoded in the mer operon which is widely distributed among prokaryotes
(Mathema et al., 2011). MerA activity was reported for At. ferrooxidans
(Olson et al., 1981; Booth and Williams, 1984) and the merA gene including
the whole mer operon was further studied (Inoue et al., 1989; Inoue et al.,
1990; Kusano et al., 1990; Velasco et al., 1999). In addition to MerA activity
another mechanism for mercury resistance has been detected in At.
ferrooxidans (Iwahori et al., 2000). This ferrous iron dependent mercury
volatilization mechanism has not been found in neutrophilic microorganisms
so far (Mathema et al., 2011) but was shown to be present in At.
ferrooxidans SUG2-2 (Sugio et al., 2001) and MON-1 (Sugio et al., 2003). In
both cases a cytochrome c oxidase is responsible for the volatilization of
mercury. However, the type aa3 cytochrome c oxidase from At. ferrooxidans
MON-1 can also volatilize mercury from organomercurials (Sugio et al.,
2010) which is not the case for cytochrome c oxidase from strain SUG2-2.
The resistance to organomercurials has not been reported before since At.
ferrooxidans does not encode the gene for organomercury lyase merB which
is responsible for this activity in neutrophiles (Mathema et al., 2011).
Metal resistance mechanisms in acidophiles have been reviewed by
Dopson et al. (2003). Mechanisms that have been well studied in acidophiles
include copper resistance (reviewed in Orell et al., 2010) and resistance to
the arsenicals, arsenate (As(V)) and arsenite (As(III)) (reviewed in Dopson et
al., 2003). Arsenic resistance is encoded in the ars operon including genes
for the As(V) reductase, arsC, the As(III) efflux pump, arsB, and the
repressor, arsR. ArsC reduces As(V) to As(III) which is subsequently
exported from the cytoplasm by ArsB. The ars operons from At.
ferrooxidans (Butcher and Rawlings, 2002), At. caldus (de Groot et al.,
2003), and Acidiphilium multivorum (Suzuki et al., 1998) have been cloned
and expressed in Escherichia coli where they conferred resistance to As(III).
Furthermore, resistance to As(V) via genes encoded by the ars operon has
been demonstrated experimentally for At. caldus (Dopson et al., 2001). In
contrast, the archaeon ‘Ferroplasma acidarmanus’ is resistant to As(V) but
it lacks a homologue of known arsenate reductase in its genome. Therefore,
it is hypothesized that a novel resistance mechanism is present (BakerAustin et al., 2007). As the ability to withstand high copper concentrations is
important for the application of acidophiles in biomining copper resistance
has also attracted large research interest. Orell and co-workers (2010)
summarize the most important acidophile copper resistance mechanisms
including: (i) many homologues of known copper resistance determinants;
(ii) multiple copies of some resistance genes; (iii) novel copper chaperones;
(iv) polyphosphate (polyP) based resistance mechanism; and (v) a defense
21
system against oxidative stress. Recently, Völlmecke et al. (2012)
demonstrated the role of the ATPases CopA and CopB as copper efflux
pumps in copper resistance of Sulfolobus solfataricus by deletion mutants.
Further, they found indications that both ATPases might be involved in
silver resistance.
In contrast to arsenical, copper, and mercury resistance the mechanisms
of acidophile zinc resistance are not well studied. Zinc is an essential metal
for most organisms. Nevertheless, it can be toxic at comparably high
concentrations. In acidophiles zinc resistance was shown to be
chromosomally encoded in At. ferrooxidans (Kondratyeva et al., 1995) and
plasmid encoded in Acidocella strain GS19h (Ghosh et al., 1997; Ghosh et al.,
2000) and Acidiphilium symbioticum (Mahapatra et al., 2002). The
resistance mechanisms itself were not elucidated but Mahapatra et al.
(2002) suggested that resistance in Acidiphilium symbioticum might be due
to an unknown mechanism.
Although resistance against metals has been studied in acidophiles the
exact molecular mechanisms still remain unknown and reports are often
limited to metal tolerances (Dopson et al., 2003; Orell et al., 2010).
However, it has been emphasized that acidophiles are generally more
tolerant to metals than neutrophiles and that this might be due to their
inside-positive membrane potential which is inversed when compared to
neutrophiles (Dopson et al., 2003; Baker-Austin et al., 2007; Franke and
Rensing, 2007; Orell et al., 2010). So far, this has not been shown
experimentally. Furthermore, care must be taken with such comparisons as
the solution chemistries of neutrophilic and acidophilic environments are
very different. Consequently, toxicity of metal ions might be very different in
both cases (as discussed above) making direct comparisons impossible.
Toxicity effects due to metal speciation or competition with other ions have
hardly or even not at all been considered in acidophile metal resistance
literature so far. Due to this and the lack of knowledge in acidophilic zinc
resistance mechanisms we studied a range of acidophiles exposed to zinc
stress. The results of this study are presented in paper III.
5. Bioenergetics
Bioenergetics is a field in biochemistry which considers the status,
generation, and flow of energy in organisms. Such considerations are
fundamental to understanding biochemical processes within any organism.
Acidophiles face particular energetic problems since they are often
challenged with low energy substrates and a very low solution pH.
Furthermore, pH homeostasis and bioenergetics are tightly connected and
pH homeostasis is central to grasp the acidophilic lifestyle. Thus, the
principles of bioenergetics will be described in the following section,
22
followed by an overview of known pH homeostasis mechanisms in
neutrophiles and acidophiles.
5.1. Principles
The prerequisite for biological energy generation is the ability of organisms
to create compartments with various concentrations of specific ions such
that their concentration gradients can be used to release energy. The main
building blocks for this energy generation are biological membranes which
constitute impermeable barriers and protons (in a few cases sodium ions)
which are used to build the concentration gradient. Thus, membranes along
with proteinaceous pumps and transporters which can bridge the border to
create or maintain the gradient are the basis of bioenergetics. Additionally,
the F1F0-ATPase enzyme complex responsible for synthesis of the central
energy currency (ATP) is of utmost importance. For prokaryotes those
energetic processes take place at the cytoplasmic membrane whereas for
higher eukaryotic systems mitochondrial or thylakoid membranes are places
of energy generation (the latter will not be considered further). Before energy
can be created the energized status of the microbial cell needs to be
established. This is achieved by primary proton pumps such as respiratory
pumps or ATP-driven proton pumps which extrude protons from the
cytoplasm establishing the proton motive force (PMF) over the membrane.
The PMF can be envisioned as a force that is acting on protons trying to pull
them across the membrane. Likewise, the PMF is an electrochemical
gradient consisting of two components: the chemical proton gradient (ΔpH)
and the membrane potential, i.e. the electrical difference in charge (ΔΨ)
(Mitchell, 1961; West and Mitchell, 1974; Booth, 1985; Macnab and Castle,
1987). Typically (for neutrophiles), the ΔpH is inside alkaline and the ΔΨ is
inside negative. The PMF is calculated according to equation (9) with R
being the gas constant, T the absolute temperature, and F the Faraday
constant:
PMF [mV] = ΔΨ – (2.3 x (RT / F) x ΔpH)
(9)
With standard conditions the formula can be simplified as in equation (10):
PMF [mV] = ΔΨ – (59 x ΔpH)
(10)
Conventionally, ΔΨ and ΔpH are calculated by subtracting the external value
from the internal value and – also by convention – the ΔΨ is negative when
the inner surface of the membrane is negative. For a functioning microbial
cell the PMF needs to be maintained at a physiological level which is around
-200 mV for most microorganisms (Krulwich et al., 2011). To achieve this
23
optimal value the two components of the PMF are adjusted to the conditions
specific for the microbe and its environment. Generally, there are typical
PMF patterns for microorganisms inhabiting different pH niches, i.e.
neutrophiles, acidophiles, and alkaliphiles (Figure 3). Neutrophiles can grow
at pH values between approximately 5.5 – 9.0 and keep an internal pH of
7.5-7.7 (Krulwich et al., 2011), whereas the cytoplasmic pH of acidophiles is
usually slightly lower at 6.0-7.0 (Cox et al., 1979). Extreme alkaliphiles, on
the other hand, grow at external pH ≥ 10 keeping a cytoplasmic pH of 7.5-8.3
(Krulwich et al., 2011). For neutrophiles the ΔpH is normally very small and
only adds a little to the PMF whereas the major contribution originates from
the large inside negative ΔΨ (Figure 3). The situation for acidophiles is
substantially different since they need to maintain a circumneutral
intracellular pH although facing a very low external pH. Thus, the ΔpH is
large and dominates the PMF. Additionally, the ΔΨ of acidophiles is
inverted, i.e. inside positive, to counteract the large negative contribution of
ΔpH. On the other hand, alkaliphiles face a different problem, i.e. their
cytoplasmic pH is more acidic than the external pH resulting in an inverted
ΔpH. To counteract the latter, alkaliphiles have a rather large negative
contribution from ΔΨ. Still, the PMF of alkaliphiles is much smaller
compared to neutrophiles and acidophiles posing specific challenges to
alkaliphiles (reviewed in Hicks et al., 2010).
Once the PMF is established work can be performed by the energy
released when protons are transferred from the high concentration
compartment (extracellular space) to the low concentration compartment
(cytoplasm). Depending on the needs of the microbial cell this work can be
ATP generation by the F1F0-ATPase enzyme complex, transporting solutes,
or flagellar movement. The F1F0-ATPase enzyme complex is ubiquitous in all
domains of life and well conserved. It is a reversible complex able to
synthesize ATP as well as hydrolyzing it coupling these reactions to the PMF
by a rotational mechanism (reviewed in Nakanishi-Matsui et al., 2010). Both
the F1 and F0 parts consist of several subunits and the F1 part of the complex
is membrane extrinsic whereas the F0 section is located within the
membrane. Much of the details of its catalytic action as well as its rotational
mechanism have been elucidated (see Nakanishi-Matsui et al., 2010 for
details). As mentioned above, some microorganisms establish a sodium
motive force (SMF) although not all of them also use the sodium gradient for
ATP generation by F1F0-ATPase (reviewed in Dimroth and Cook, 2004). For
example, some anaerobic microorganisms, e.g. Propionigenium modestum,
or Acetobacterium woodii devise a special type of F1F0-ATPase coupled to
the SMF for ATP production (reviewed in Dimroth and Cook, 2004). In
contrast some marine species like Vibrio spp. are exposed to high salt
contents and utilize primary sodium pumps to extrude sodium from the
cytoplasm (Hayashi et al., 2001). Nevertheless, those species utilize the PMF
24
for ATP generation (Krumholz et al., 1990) but can harness the energy from
the SMF to import nutrient and drive flagellar movement (Yorimitsu and
Homma, 2001).
Figure 3. Proton motive force (PMF) in acidophiles, neutrophiles, and alkaliphiles. Summary (A) of
membrane potential (ΔΨ), pH gradient (ΔpH), and cytoplasmic pH measured for the acidophiles At.
ferrooxidans (Cox et al., 1979) and A. acidocaldarius (Michels and Bakker, 1985); the neutrophiles E. coli
(Zilberstein et al., 1979) and B. subtilis (Shioi et al., 1978); as well as the alkaliphiles B. pseudofirmus (Sturr et
al., 1994) and B. alcalophilus (Guffanti et al., 1978). The figure is redrawn from the original references and a
review in the field (Krulwich et al., 2011). It exemplifys typical PMF patterns for microorganisms inhabiting
three different pH niches (acidic, neutral, and alkaline). Schematic overview (B) of acidophile At.
ferrooxidans, neutrophile E. coli, and alkaliphile B. pseudofirmus showing typical values for ΔpH and ΔΨ as
well as the resulting PMF.
5.2. Mechanisms for pH Homeostasis
As shown in the previous section, ΔpH is directly connected to the energy
status of the microbial cell. In the case of acidophiles the ΔpH is of even
greater importance for bioenergetics as it is the only productive component
of the PMF. Not only because of its implications for bioenergetics but also
due to fundamental physiological consequences control of internal pH is
crucial for all organisms. Acidification of the cytoplasm leads to protonation
of biomolecules and thus, e.g. to the denaturation of proteins. Hence, the
control and maintenance of the internal pH, i.e. pH homeostasis, is a basic
requirement for survival (reviewed in Slonczewski et al., 2009; Krulwich et
al., 2011).
25
Mechanisms of pH homeostasis have been studied quite extensively in
neutrophiles, especially in E. coli (reviewed in Foster, 2004) and
Helicobacter pylori (Sachs et al., 2011) since both species can survive
intermittently at very low pH. In comparison, little is known about pH
homeostasis in acidophiles (reviewed in Baker-Austin and Dopson, 2007). A
possible way to counter excess protons entering the microbial cell is
cytoplasmic buffering. Several molecules in the cell can act as buffering
substances, e.g. amino acids or the amino acid side chains of proteins,
polyamines, inorganic phosphate, and polyP. Apart from being buffering
substances polyamines have also been shown to fulfill other roles in acid
stress response of E. coli, such as blockage of outer membrane porins
(Samartzidou et al., 2003). Whether a certain molecule can provide
buffering capacity at a specific pH depends on its pKa value. Phosphate and
polyP have a pKa of 7.2 making them more suitable for cytoplasmic buffering
of neutrophiles than acidophiles. For acidophiles protein amino acid side
chains are more likely responsible for buffering as they offer a large range of
pKa values (Zychlinsky and Matin, 1983). Cytoplasmic buffering capacity
exists in all microorganisms, though levels of the capacity varies between
species (Krulwich et al., 1985) and it was questioned whether this
mechanism is part of the global pH homeostasis in alkaliphilic bacilli
(Krulwich et al., 1985). However, it was shown that the buffering capacities
of the neutrophile E. coli and two acidophiles (A. acidophilum and bacterium
PW2) are comparable and effective in supporting a constant internal pH
upon influx of protons (Zychlinsky and Matin, 1983; Goulbourne et al.,
1986).
Although passive cytoplasmic buffering can support pH homeostasis it is
believed that active mechanisms are more important. For neutrophiles as
well as acidophiles active transport of protons either via primary proton
pumps or secondary cation/proton antiporters is of great importance for the
maintenance of a stable internal pH (Baker-Austin and Dopson, 2007;
Slonczewski et al., 2009; Krulwich et al., 2011). Respiratory chain primary
proton pumps were studied in At. ferrooxidans (Chen and Suzuki, 2005;
Ferguson and Ingledew, 2008), ‘F. acidarmanus’ (Dopson et al., 2005), as
well as in several archaeal acidophiles (reviewed in Schäfer et al., 1999).
Interestingly, the At. ferrooxidans cytochrome c involved in ferrous iron
oxidation differs from homologues in neutrophiles as it lacks one of the
putative proton channels (Ferguson and Ingledew, 2008). Further,
secondary cation/proton antiporters have an important role in modulating
the PMF, e.g. the E. coli Na+/H+ antiporter NhaA is essential for adaptation
to alkaline stress (Padan et al., 1989). Additionally, it has been perceived that
potassium ions might play an important role for acidophile pH homeostasis
possibly by establishing the inside positive ΔΨ. Until now this has not been
shown unequivocally but circumstantial experimental data as well as data
26
based on genomic sequences support this hypothesis. In At. thiooxidans it
was demonstrated that potassium ions were the most efficient cations tested
to maintain the ΔΨ (Suzuki et al., 1999). Further, it was shown that
Sulfolobus spp. required the presence of potassium ions during respiratory
proton pumping (Schäfer et al., 1996). Based on sequence data it was found
that Leptospirillum group II (Tyson et al., 2004), Picrophilus torridus
(Fütterer et al., 2004), and Sulfolobus solfataricus (She et al., 2001) encode
a large number of putative secondary cation transporters possibly involved
in creation and maintenance of the ΔΨ.
A prevalent mechanism during acid or base stress in neutrophiles is the
expression of acid or base consuming enzymes (Foster, 2004; Slonczewski et
al., 2009). A well-studied system essential for E. coli acid stress survival is
the decarboxylase system (reviewed in Foster, 2004) but other species, such
as lactococci and bacilli, have also been shown to express acid dependent
decarboxylases (reviewed in Slonczewski et al., 2009). For example, the
glutamine decarboxylases GadA and GadB in E. coli catalyze the
decarboxylation of glutamine which is a proton consuming reaction. The end
products are carbon dioxide and γ-amino butyric acid (GABA). The
decarboxylases are co-expressed with an antiporter, GadC, which exports
GABA in exchange for glutamine (Lin et al., 1995; Hersh et al., 1996; Richard
and Foster, 2004). Similar systems are available for the acid dependent
decarboxylation of arginine, lysine, or histidine, amongst others.
Comparable acid resistance systems have not been detected in acidophiles to
date. This might not be surprising as acidophiles typically inhabit
environments scarce of organic compounds unlike neutrophiles which can
easily import amino acids as substrate for proton consuming
decarboxylation. The same system would probably require much more
energy for acidophiles due to the necessity to synthesize large amounts of
amino acids. However, when it comes to other enzymatic reactions which
might be involved in protection against acid it is interesting to note the
ability of chemoorganotropic acidophiles to degrade organic acids. Organic
acids are extremely toxic to acidophiles (Borichewski, 1967; Tuttle and
Dugan, 1976; Aston et al., 2009) as they are protonated and neutral at the
external low pH and can easily enter the cell. Within the cytoplasm they
rapidly dissociate due to the prevailing circumneutral pH releasing protons
and acidifying the cell (Ingledew, 1982; Alexander et al., 1987). It has been
suggested that the ability to degrade organic acids by extremely acidophilic
species, such as Ferroplasma spp. (Dopson et al., 2004) or Picrophilus spp.
(Ciaramella et al., 2005), might be part of their pH homeostasis system.
Reorganization of the cytoplasmic membrane in response to acid
challenge has been reported for neutrophiles (Chang and Cronan, 1999) as
well as acidophiles (Mykytczuk et al., 2010). Studies on E. coli have
demonstrated the involvement of cyclopropane fatty acids in the low pH
27
response (Chang and Cronan, 1999). Furthermore, the microbial sterol
analogues, hopaniods, are involved in acid stress in neutrophiles (reviewed
in Kannenberg and Poralla, 1999). While Mykytczuk et al (2010) studied the
fatty acid profiles of several strains of At. ferrooxidans and showed that the
membrane fluidity was adjusted in response to external pH in a strainspecific manner. The importance of rigid archael membranes composed of
tetraether lipids for inhabiting extreme environments has been pointed out
on many occasions (van de Vossenberg et al., 1998; Albers et al., 2000;
Konings et al., 2002; Baker-Austin and Dopson, 2007). Additionally,
changes due to low pH have been detected in the At. ferrooxidans outer
membrane (Amaro et al., 1991). Particularly, the At. ferrooxidans OMP40
outer membrane pore has specific characteristics, such as small pore size and
amino acid composition, which suggest an acid adaptation of the protein
(Guiliani and Jerez, 2000). Unfortunately, this is the only report on the
subject and it would be interesting to confirm such acid adaptation for
further acidophile outer membrane proteins (OMPs). Finally, it has been
proposed that the inside positive membrane potential is an intrinsic
mechanism of acidophiles to hamper the leakage of protons into the
cytoplasm (Baker-Austin and Dopson, 2007). Due to this lack of knowledge
in acidophilic pH homeostasis the proteomic response to growth in nonoptimal pH conditions was studied in At. caldus with paper IV.
6. The Post-Genomic Era
Fifteen years ago the genome sequence of E. coli, the most studied
microorganism and one of molecular biology’s workhorses, was announced
(Blattner et al., 1997) and about ten years ago the human genome was
successfully sequenced (Venter et al., 2001). These were the beginnings of
vast genome sequencing endeavors which resulted in an exponentially
growing amount of sequence data from all domains of life. The technical
advances during the last 10 years have also allowed for further new, global
methodologies such as proteomics and metabolomics which let us into the
so-called post-genomic era. By integrating the various approaches with
systems biology, those developments opened the door ajar to glimpse at the
big question of what life is. In the following sections the toolkit (genetic
tools, ‘omics’ techniques, and use of continuous culture) of the post-genomic
era will be explained and put into context with acidophile research. In the
end of this section, special notice is given to systems biology with its
daunting tasks and its great potential.
28
6.1. Genetic Tools for Acidithiobacilli
An important tool in molecular biology research is the genetic manipulation
of organisms primarily used for evaluating the function of unknown genes.
Creating knock-out mutants of the genes in question followed by phenotypic
testing and evaluation of the complemented mutant is an elegant way to
assess a hypothesized function. The function can be further characterized by
cloning the target genes and e.g. heterologous expression of the
corresponding proteins in a host such as E. coli followed by subsequent
purification. Targeted point mutations or partial deletions can help to study
the structure or function of certain domains within the protein of interest.
Genetic manipulations of acidophiles, however, have been difficult to realize
probably due to their distinct physiology and because the available genetic
protocols are optimized for pH neutral conditions (Holmes and Bonnefoy,
2007). Only few reports of successfully created mutants of acidithiobacilli
have been published and no standard protocols are available. Major
obstacles for an efficient method are, apart from the introduction of
exogenous DNA, long generation times and poor growth on solid media
which makes enrichment and selection of mutants extremely cumbersome.
The most common technique for introducing exogenous DNA to neutrophilic
organisms is electroporation (also termed electrotransformation).
Remarkably, there are only three reports of electroporation applied to
acidothiobacilli. In 1992 Kusano et al. (1992) reported the electroporation of
At. ferrooxidans, however, the protocol was not very efficient with about 200
transformants per µg of DNA. Furthermore, the protocol did not work on all
the strains tested. Recently a more efficient protocol for At. caldus was
presented (Chen et al., 2010) where a maximum transformation efficiency of
3.6 x 104 transformants per µg DNA was achieved. The authors introduce
this as a convenient and robust protocol and suggest the usage as a standard
protocol for recombinant gene transfer and creating knock-out mutants. In
their study they only show a proof of principle by introducing marker genes
in the At. caldus genome without demonstrating a knock-out mutant nor
testing several recipient strains. Nevertheless, more recently, the same
authors successfully applied their protocol for construction of a sulfur
oxygenase reductase (Sor) deletion mutant (Chen et al., 2012). Another
method for genetic manipulation is conjugation which was applied for most
acidithiobacilli (see Table 4 for an overview and references). In most studies
the maximum conjugation frequencies were around 10-4 and 10-5
transconjugants per recipient. Though, frequencies were often not very
reproducible with large variations for the same protocol (van Zyl et al.,
2008), depending on the plasmid (Jin et al., 1992; Liu et al., 2000; Liu et al.,
2007), or depending on the recipient strain (Liu et al., 2000). Mostly
conjugation was applied to introduce a heterologous gene, such as a metal
29
resistance gene, to genetically modify the recipient strain (Table 4). Only in
three reports conjugation was used to create deletion mutants (Liu et al.,
2000; van Zyl et al., 2008; Wang et al., 2012) followed by functional
assessment of those genes. In both At. ferrooxidans (Liu et al., 2000), and
At. caldus (van Zyl et al., 2008), marker exchange was used to disrupt the
target genes and in both cases a kanamycin resistance marker gene was
applied. In contrast, a markerfree approach based on the homing
endonuclease I-SceI was used to create a knock-out of putative
phosphofructokinase in At. ferrooxidans (Wang et al., 2012). Another recent
report demonstrated the applicability of a small broad-host-range vector,
pBBR1MCS-2, for efficient conjugation of acidithiobacilli (Hao et al., 2012).
Unlike vectors previously used for conjugation pBBR1MCS-2 does not belong
to the IncQ, IncW or IncP groups. The new plasmid proved to be efficiently
transferred and stable and might be a useful future genetic tool (Hao et al.,
2012).
Table 4. Overview of conjugation protocols for acidithiobacilli
Method
Trait studied
Max.
Transfer
frequency
Reference
At. ferrooxidans
heterologous gene
transfer
arsenic resistance
2.9 x 10-4
Peng et al., 1994a
At. ferrooxidans
marker exchange,
knock-out mutant
recA gene
10-3
Lui et al., 2000
At. ferrooxidans
markerless gene
replacement,
knock out mutant
phosphofructokinase
10-4
Wang et al., 2012
arsenic resistance
10-4
Zhao et al., 2005
arsenic resistance
n.a.a
Kotze et al., 2006
proof of principle
10-4
Lui et al., 2007
arsenic resistance,
tetrathionate
hydrolase
10-5
van Zyl et al., 2008
mercury resistance
1.5 x 10-4
Chen et al., 2011
proof of principle
10-7–10-5
Hao et al., 2012
proof of principle
10-5
Jin et al., 1992
proof of principle
10-5
Peng et al., 1994b
phosphofructokinase
10-5
Tian et al., 2003
Organism
At. caldus
At. caldus
At. caldus
At. caldus
At. caldus
At. caldus and At.
thiooxidans
At. thiooxidans
At. thiooxidans
At. thiooxidans
a n.a.
heterologous gene
transfer
heterologous gene
transfer
heterologous gene
transfer
marker exchange,
knock-out mutant
heterologous gene
transfer
heterologous gene
transfer
heterologous gene
transfer
heterologous gene
transfer
heterologous gene
transfer
Not possible to determine the transfer frequency as distinct colonies were not
formed.
30
Molecular cloning of genes and their heterologous expression in a host
such as E. coli is technically more straight forward and has been reported for
genes of acidithiobacilli even prior to the availability of the respective
genome sequence (Barros et al., 1985; Shiratori et al., 1989). For periplasmic
and OMPs which are naturally exposed to acidic pH it has been reported that
heterologous expression in a neutrophilic host can lead to false folding or
failure of co-factor binding (Bengrine et al., 1998; Kanao et al., 2010).
Interestingly, the heterologous expression of a periplasmic protein secreted
though the twin arginine translocation (tat) pathway yielded a protein with
properly bound co-factor (Bruscella et al., 2005). This is probably due to the
fact that in the tat system proteins are fully folded within the cytoplasm and
not in the periplasm, as it is the case for the secretory (sec) pathway
(Bruscella et al., 2005).
In conclusion, almost 20 years of efforts in establishing a method for the
genetic manipulation of acidithiobacilli has so far not lead to routine use of
any of the suggested protocols. Possibly, recent developments such as the
protocols of Chen et al. (2010), Hao et al. (2012), or Wang et al. (2012)
might establish themselves as standard procedures. In any case, a robust and
convenient protocol would greatly advance the basic and applied research in
acidophiles. Until such routine genetic analysis is available the use of ‘omics’
(Burg et al., 2011) techniques will continue to provide useful insights into
acidophile physiology and community interactions as will be discussed in the
following section.
6.2. ’Omics‘ Techniques and Continuous Culture
All ‘omics’ techniques are global approaches which seek to depict the entire
collection of a certain biomolecule present under a specific condition and
time (Figure 4), i.e. genomics is the collection of all genes encoded by an
organism, transcriptomics captures all RNA transcripts of active genes and
so on. Initially, all ‘omics’ techniques pertained to single organisms and pure
strains, however, it is also possible to study much more complex microbial
communities with the same approaches. Usually the prefix ‘meta’ or the
terms ‘community’ and ‘environmental’ are used to denote this application
(Figure 4).
In recent years DNA sequencing technologies have dramatically improved
while costs have dropped which allows fast and inexpensive sequencing of
genomes or metagenomes. The traditional Sanger DNA sequencing method
is now largely replaced by high throughput next generation sequencing
(NGS) approaches (Novais and Thorstenson, 2011; Shokralla et al., 2012).
Several commercial NGS platforms are available having individual
advantages and drawbacks (Shokralla et al., 2012). One example is a PCRbased technology for short- to medium-reads termed pyrosequencing which
31
functions by detecting the release of pyrophosphate during DNA synthesis
(Novais and Thorstenson, 2011).
Today the genome sequences of all acidithiobacilli except At. albertensis
are available (Valdes et al., 2008b; Valdes et al., 2009; Liljeqvist et al., 2011;
Valdes et al., 2011). In the first place the availability of the genome
sequences enabled in silico analysis of the predicted genes and gene products
thereby providing insight into the genetic potential of the organism (Valdes
et al., 2008b; Quatrini et al., 2009). Furthermore, with the availability of
several acidophilic genome sequences it was possible to compare
acidithiobacilli with each other (Valdes et al., 2008a) or specific traits within
the acidophile community such as carbon and nitrogen fixation (Levican et
al., 2008). Such bioinformatic analyses of several genome sequences is
named comparative genomics and it can aid in assessing the genetic
capacities of organisms and provide clues on interesting traits which should
be studied in more detail. An overview of available acidophile genome
sequences and associated bioinformatics studies is given in a recent review
by Cardenas et al. (2010). But not only are the genome sequences of pure
strains rewarding but also genome sequences of the whole microbial
community in a certain environmental niche are of great interest. These
sequences are commonly called metagenomes. The immense benefit that
metagenomics holds is that it can capture all members of a microbial
community even those that are uncultivable (reviewed in Simon and Daniel,
2011). The accessibility of uncultivable microbes has already allowed the
discovery of new antibiotics and enzymes and will undoubtedly further
provide impetus to biotechnology (reviewed in Schloss and Handelsman,
2003). Additionally, metagenomics can also advance our knowledge in
microbial ecology, as well as evolution and diversity (reviewed in RodriguezValera, 2004). However, with the ability to sequence metagenomes new
challenges are connected which have been pointed out recently by several
researchers (Desai et al., 2012; Dini-Andreote et al., 2012). Their concerns
are largely related to computational problems due to large amounts of data
and the difficulty to resolve complex community structures on a strain level
(Desai et al., 2012; Dini-Andreote et al., 2012). A milestone in metagenomic
studies was the random shotgun sequencing analysis of an AMD community
from the Iron Mountain Mine, USA, which revealed the near-complete
genome sequences of Ferroplasma type II and Leptospirillum group II as
well as other partial genomes (Tyson et al., 2004). The low diversity within
the AMD community allowed effective genome reconstruction. With the help
of the genomic data, information about prevailing metabolic pathways and
survival strategies in acidic environments was reconstructed. Recently,
pyrosequencing has been applied to study the metagenome of acidic
biofilms, called ‘snottites’ that form in hydrogen sulfide rich caves (Jones et
al., 2012). Although there are major geochemical differences between the
32
snottite habitat and AMD sites both acidophilic communities showed
remarkable similarities: (i) low microbial diversity; (ii) dominance of
acidophilic chemolithoautrotrophs; (iii) lower contribution of heterotropic
microbes; and (iv) membrane adaptations to survival in acidic environments
(Jones et al., 2012).
Figure 4. Overview of ‘omics’ techniques and systems biology. All ‘omics’ techniques can be applied for pure
cultures and microbial communities investigating various aspects of organisms or environmental
communities. Data generated by ‘omics’ techniques can be used to generate mathematical models of biological
systems. The models can be used to test hypotheses about the systems and can further be refined in an
iterative way.
Although genome sequences of pure strains as well as metagenomes
largely expanded our knowledge of acidophiles and their communities, the
type of information available from DNA sequences is limited to putative
function only. In order to receive functional information other types of
analysis such as transcriptomics and proteomics are required. As
transcriptomics is based on nucleotide sequencing the same sequencing
methods as for genomics and metagenomics can be applied. The first
approaches to transcriptomics were done by extraction of mRNA and reverse
transcription into DNA. Analysis was frequently done with microarrays
bearing probes for genes of interest usually covering the whole genome
(reviewed in Watson et al., 1998). The latest technique is to directly sequence
the transcripts using NGS techniques. In contrast to DNA sequences,
transcriptomics is based on mRNA sequences and therefore, gene expression
information is retrieved. By using microarrays and mRNA extractions from
33
acidophilic microorganisms grown at different conditions differential gene
expression profiles can be accessed and used to learn more about stress
responses (Yin et al., 2012), metabolic pathways (Appia-Ayme et al., 2006;
Quatrini et al., 2006; Auernik and Kelly, 2008), and also environmental
community functions by metatranscriptomics (Parro et al., 2007; MorenoPaz et al., 2010).
Proteomics takes functional analysis one step further by analyzing the
whole set of proteins expressed in a certain condition or environment
(metaproteomics/community proteomics). Proteins are non-homogenous
molecules having diverse properties, differing in e.g. charge, hydrophobicity,
molecular weight (MW), and a large dynamic range of abundance. This
complexity makes it difficult to simultaneously analyze all proteins expressed
in a certain condition using the same method. Thus, a variety of different
methods was developed targeting various subproteomes and protein species
thereby attempting to circumvent this problem (Poetsch and Wolters, 2008;
Casado-Vela et al., 2011; Ly and Wasinger, 2011). On the other hand,
proteomics has a technical advantage over transcriptomics as proteins are
more stable than mRNAs thus analysis is more robust. Furthermore,
proteins are in most cases the final, functional gene products (although there
are exeptions such as genes coding for small regulatory RNA for example)
making information derived from proteomics a very strong case. Moreover,
it has been noted in many studies trying to correlate transcriptomics and
proteomics data that mRNA levels do not always correspond to protein levels
which makes it extremely difficult to extrapolate functional information from
transcriptomics data alone (Pradet-Balade et al., 2001 and references
within). The two main approaches in proteomics are gel-based and gel-free
methods. The most common gel-based method is two dimensional
polyacrylamide gel electrophoresis (2D-PAGE) which can visualize and
quantify the protein spots directly on a gel and subsequently spots of interest
are identified by mass spectrometry (MS). The disadvantage of this approach
is the incompatibility with integral membrane proteins and its limited
dynamic range. On the contrary, gel-free approaches mostly based on the
mass spectrometric analysis of peptide mixtures in solution have been shown
to detect membrane proteins. Both methodologies have strengths and
weaknesses, however when it comes to quantification of expression levels the
‘traditional’ 2D PAGE approach still has advantages over quantification by
MS (Wilmes and Bond, 2006). Generally, the identification of proteins is
much more straight forward if genome data of the studied organism is
available. If this is the case most proteins can easily be identified by Matrix
assisted laser desorption ionization Time of Flight (Maldi-ToF) MS. If no
sequence data is available cumbersome de novo sequencing MS techniques
have to be used. Gel-based differential expression proteomics was applied to
study various aspects of acidophile physiology. Early work on At.
34
ferrooxidans included differential proteomics in response to pH stress
(Amaro et al., 1991), phosphate starvation (Vera et al., 2003), and different
energy substrates (Ramirez et al., 2004). Additionally, periplasmic proteins
were investigated using a gel-free approach (Chi et al., 2007). Gel-based
proteomics was also used to help elucidate aspects of Ferroplasma spp.
physiology such as metal resistance (Baker-Austin et al., 2005; Baker-Austin
et al., 2007), anaerobic growth (Dopson et al., 2007), and iron homeostasis
(Potrykus et al., 2011). In contrast to the investigation of a single organism,
metaproteomics or community proteomics provide the opportunity to gain
functional insights into the whole microbial community of a given habitat
(reviewed in Wilmes and Bond, 2006; Wilmes and Bond, 2009). The first
report of metaproteomics of an acidophilic community was conducted on
biofilm samples from AMD sites at Iron Mountain, USA (Ram et al., 2005).
The authors used a gel-free, shotgun MS technique as well as metagenomic
data for their analysis. They revealed that apart from anticipated metal and
acid stress, oxidative stress also plays a role in AMD sites. Further, it was
shown that certain members of the community were responsible for specific
processes, such as extracellular polymer production or nitrogen fixation
(Ram et al., 2005). Other studies of AMD communities also combined
metagenomic and metaproteomic data (Lo et al., 2007; Goltsman et al.,
2009). Additional work on AMD communities in the laboratory of Jillian
Banfield advanced those ‘omics’ approaches by integrating proteomics,
geochemical, and biological data of multiple communities and over a period
of time (Mueller et al., 2010; Mueller et al., 2011). In the same research
group a method for cultivation of AMD biofilms under laboratory conditions
was developed which can now be used to test hypotheses under controlled
conditions (Belnap et al., 2010; Belnap et al., 2011). Certainly, integrated
‘omics’ approaches such as those mentioned will further help to understand
the complex processes and interactions within environmental communities
(reviewed in Zhang et al., 2010).
One of the most recent ‘omics’ methods is the investigation of the entire
set of metabolites under a given condition (Figure 4), termed metabolomics.
Metabolomics is a potent technique which is capable of quantitatively
analyzing hundreds of metabolites in one sample. Often the samples are
separated by liquid chromatography and subsequently analyzed by MS.
Additionally, nuclear magnetic resonance or gas chromatography coupled to
MS (GC-MS) are possible alternative methods for analysis. Due to the vast
complexity and the possibly very short half-lives of metabolites existing in a
microbial cell this type of analysis bears its specific challenges. However,
with advancing analytical techniques those challenges are being addressed
(Patti et al., 2012). Metabolomics expands the functional studies of
transcriptomics and proteomics to yet another level and additionally allows
information on pathway regulation and metabolite fluxes (termed fluxomics)
35
to be collected. When metabolomics is applied to communities of several
organisms it is termed environmental metabolomics which is another
emerging field (reviewed in Viant, 2008; Bundy et al., 2009). Recently, the
first metabolomics study on acidophiles has been published (Martínez et al.,
2012). Strains of At. ferrooxidans and At. thiooxidans were grown on
various substrates and their metabolic profiles investigated using capillary
electrophoresis and MS. The polyamide spermidine was detected in both
strains and the authors suggest a role in biofilm formation whereas the
finding that glutathione was present primarly in the sulfur oxidizing species
strengthened its suggested role as activator of sulfur during the sulfur
oxidation pathway (Martínez et al., 2012). Another interesting approach is
the application of metabolomics to study toxicity (reviewed in Booth et al.,
2011) which might also be of interest for metal toxicity in acidophiles.
In order to draw valid conclusions from ‘omics’ experiments (as for other
biological experiments) the experimental design is of great importance. For
the study of pure cultures and model organisms the cultivation of
microorganisms in the laboratory is obligatory. Consequently, the
experimental design starts with growing the biomass for the experiments.
The method of culturing will unavoidably influence the physiology of the
organism under study. Thus, the apparently trivial task of growing the
biomass for a subsequent experiment turns out to be an essential part of the
experimental design. Fundamentally, there are two types of liquid culturing
techniques available: (i) the closed system, i.e. batch cultures; and (ii)
continuous cultures which are open systems (Figure 5). Often batch cultures
are the routine mode of growth in many microbiological laboratories since
they are simple and do not require special equipment or maintenance. A
certain amount of medium containing all substances necessary for growth is
inoculated with microorganisms, then placed at the desired temperature and
usually aerated by shaking. During this mode of growth the bacteria
continuously adapt to varying conditions as substrates are used up while
metabolites and waste products accumulate in the medium (Figure 5A). This
is often accompanied by changes in pH and possibly other parameters (such
as redox potential in the case of iron rich media for acidophilic iron
oxidizers). Those changing conditions are avoided in the open system where
a constant culture volume is kept in balance by continuously feeding fresh
growth medium and removal of superfluous culture liquid (waste, Figure
5B). After inoculation and equilibration of the continuous culture the
microorganisms grow at a fixed, constant rate and all concentrations within
the culture are constant as well (Figure 5B). Therefore, the continuous mode
of growth avoids the largest disadvantage of batch culturing, i.e. changing
growth phases which can possibly mask the physiological response being
studied.
36
Recently, microbiologists have rediscovered the benefits of continuous
cultures and realized that in order to address the complex biological
questions of today culturing microorganisms in a strictly controlled manner
is a prerequisite (Hoskisson and Hobbs, 2005; Pullan et al., 2008; Bull,
2010). Especially, when it comes to applying ‘omics’ techniques samples
originating from continuous growths are better suited (Hoskisson and
Hobbs, 2005; Pullan et al., 2008), showing for example less variation (Piper
et al., 2002). Furthermore, a recent study demonstrated that the toxicity of
metals differs significantly for bacteria grown in batch and continuous
culture (Şengör et al., 2012) which is important to consider for research and
application of acidophiles as they are often faced with high concentrations of
metals.
Figure 5. Batch (A) and continuous (B) systems for culturing microorganisms. Batch cultures are closed
systems where generation time/biomass of microorganisms as well as concentrations of substrate and
metabolites change over time. In continuous cultures a constant volume is kept by feeding fresh substrate,
and removing waste, thereby constant concentrations of biomass, substrate, and metabolites as well as a fixed
generation time are maintained.
6.3. Systems Biology
Systems sciences are multidisciplinary approaches to complex systems. Data
from parts comprising the system of interest are collected, studied, and
evaluated. Finally, mathematical tools are used to infer a model describing
37
the system. This model can be used to make predictions of the system and by
testing the model with a new set of data it can subsequently be improved in
an iterative way (Figure 4). The great potential of the systems sciences is that
they integrate all aspects of the system, and thereby leave the reductionist
point of view behind. Systems sciences are for example thermodynamics or
ecology (Smith et al., 2012). However, with the advent of ‘omics’ techniques
and the large amounts of data that are produced by applying them, systems
sciences are also being applied to biological systems (Ideker et al., 2001;
Kitano, 2002b, a), and the term systems biology was coined. Systems biology
can describe processes at such a small scale as a single cell or expand to
larger scales such as a multi-cellular organism or a whole habitat. Since its
emergence, systems biology has been praised as the key to understand the
principles of life. Although we are still at the beginning of this quest, which
started about 10 years ago, scientists still believe that systems biology can
provide those answers (Pfau et al., 2011; Ideker and Krogan, 2012; Khatri et
al., 2012). Undoubtedly, many challenges still lie ahead but progress has
been made (Pfau et al., 2011; Ideker and Krogan, 2012; Khatri et al., 2012). A
recent review on systems biology of the model microbe E. coli describes how
genome scale models can be applied to five categories: (i) metabolic
engineering; (ii) bacterial evolution; (iii) network analysis; (iv) phenotypic
behavior; and (v) biological discovery (Feist and Palsson, 2008). The
research of lactic acid bacteria has also benefited from systems biology
approaches by providing “new knowledge or perspective, often
counterintuitive, and clashing with conclusions from simpler approaches”
(Teusink et al., 2011). Another interesting review highlights the benefits of
systems biology approaches for the study of toxicity, including metal
mixtures (Spurgeon et al., 2010). When it comes to metal-microbe
interactions two studies are of particular interest, a constraint based model
on Geobacter sulfurreducens (Mahadevan et al., 2006) and dynamic multispecies metabolic modeling (DMMM) on Geobacter and Rhodoferax
(Zhuang et al., 2011). Geobacter sulfurreducens grows anaerobically by
oxidation of organic compounds coupled to the reduction of ferric iron and it
can be applied for the remediation of metal or organic loaded water. With
the help of the constraint based model, Mahadevan and co-workers (2006)
explored the central metabolism and electron transport of G. sulfurreducens.
They point out that the iterative modeling together with experimentation can
aid in comprehending the physiology of poorly studied organisms which are
of environmental interest and eventually this can help in improvement of
their application. A great advance for the study of microbial community
interactions is the report on the first multiple genome scale metabolic model
(Zhuang et al., 2011). Here the DMMM is used to predict the microbial
interactions of two ferric iron reducing organisms which are competing in in
situ bioremediation of ground water contaminated with uranium. Although
38
the presented model takes only two members of the microbial community
into account this approach has significant potential for future studies of
microbial ecology. Also in acidophile research mathematical metabolic
models based on genome sequence data have been developed. So far
stoichiometric models of At. ferrooxidans (Hold et al., 2009) and L.
ferrooxidans (Merino et al., 2010) have been presented and both showed
good behavior when comparing calculated values with experimental data
from literature. Certainly, more such approaches are needed to advance our
knowledge of acidophile physiology and community structure.
39
Aims of This Study
The main aim of this study was to elucidate strategies of acidithiobacilli to
cope with the extreme conditions prevailing in their typical habitat. In order
to meet this aim several aspects of acidithiobacilli physiology were
investigated in more detail: (i) substrate metabolism; (ii) metal resistance;
and (iii) pH homeostasis.
At. caldus is an important sulfur oxidizer within the acidophile community
and valuable for industrial biomining. Still its sulfur oxidation pathways are
poorly characterized. In paper I we aimed to learn about sulfur metabolism
in At. caldus.
A lot is known about At. ferrooxidans sulfur and iron oxidation. However,
the metabolic pathways for anaerobic sulfur oxidation coupled to ferric iron
respiration are still elusive. We aimed to elucidate those pathways and find
the enzyme responsible for ferric iron reduction in paper II.
Zinc can reach high concentrations in AMD environments or biomining
activities. Such high concentrations of this essential metal ion can be toxic.
With a comparative study on three acidophilic model organisms we aimed to
clarify the mechanisms of zinc tolerance in model organisms in paper III.
The main characteristic of acidophiles is their acidophilic nature which is yet
poorly understood. In paper IV we aimed to learn more about pH
homeostasis in At. caldus.
40
Results and Discussion
In order to reach the specific aims four studies dealing with the individual
aspects have been conducted and are presented within this thesis. As
reviewed in chapter 6 of the introduction, tools for genetic manipulation of
acidophiles are still not feasible for routine experimentation whereas various
’omics’ techniques offer great potential for the study of extremophiles.
Furthermore, the recent availability of the At. caldus genome sequence
offered new possibilities for the investigation of this organism, such as
bioinformatics and proteomics. Consequently, a focus of this thesis was to
apply ‘omics’ techniques, mostly proteomics, to study acidophilic model
organisms. The proteomics experiments were based on differential protein
expression assessed by 2D-PAGE and protein identification by Maldi-ToF. In
the following the results of the presented papers are summarized and their
contribution to the field discussed.
Paper I: Sulfur metabolism in the extreme acidophile
Acidithiobacillus caldus
Previous knowledge of At. caldus sulfur oxidation pathways was fragmentary
despite the importance of this organism for industrial applications.
Consequently, the newly available genome sequence of At. caldus was
scrutinized by bioinformatics to predict genes putatively involved in sulfur
metabolism. As the bioinformatics survey revealed a different set of sulfur
oxidation enzymes compared to At. ferrooxidans a more detailed study was
initialized. For the following experiments At. caldus was grown in
continuous culture on two different sulfur compounds, solid elemental sulfur
and soluble tetrathionate. The first indication that putative sulfur oxidation
genes were activated under these conditions was provided by measuring
mRNA levels of selected genes with semi-quantitative reverse transcription
polymerase chain reaction (semiQ RT-PCR). Furthermore, a proteomics
approach was used to pinpoint differentially expressed proteins during the
utilization of the two sulfurous substrates. Based on the results, a model for
sulfur oxidation in At. caldus was presented.
An overview of the genes putatively involved in sulfur metabolism
revealed by the bioinformatics analysis is presented in Figure 1A of paper I.
Interestingly, At. caldus harbors homologous genes detected in At.
ferrooxidans as well as additional genes which have not been previously
shown in acidithiobacilli. The former includes the genes sqr, tth, and hdr,
whereas the latter were the sulfur oxidation (sox) genes and sor. Except for
tth and sor, two copies of the respective genes were detected in the At. caldus
genome. Most striking was that two sox gene clusters with differing gene
41
organization were found. Both of them contained soxABXYZ which together
encode a protein complex required to oxidize thiosulfate. Thiosulfate
oxidation by the Sox system has been well studied in Paracoccus
pantotrophus (Friedrich et al., 2005) and green sulfur bacteria (Sakurai et
al., 2010) and the presented paper is the first report of a Sox system in
acidophiles. The enzyme Sor, on the other hand, has been studied in the
archaea Acidianus ambivalens (Urich et al., 2004) and its activity has been
demonstrated in At. caldus like strains (Janosch et al., 2009).
Based on the bioinformatics predictions primers were designed
specifically targeting a selection of the putatively involved genes. The results
of semiQ RT-PCR for At. caldus grown on tetrathionate and solid sulfur are
shown in Figure 2 of paper I. All sox genes except soxZ from both gene
clusters were up-regulated during the growth on tetrathionate and no
detectable differential expression was noted with any of the other genes
tested. These results suggested that both sox clusters were regulated alike
and were possibly involved in thiosulfate oxidation which is a degradation
product of tetrathionate (de Jong et al., 1997). The reason why both soxZ
copies did not follow the same trends as the other sox genes remains
unknown. Additionally, no differential mRNA levels for sor implied that this
enzyme was transcribed at similar levels during the conditions tested. The
same was true for tth and doxD which was unexpected as they have been
previously reported to be up-regulated upon growth on tetrathionate
(Rzhepishevska et al., 2007).
The proteomics study of this paper was divided into two comparisons.
Firstly, continuous culture samples grown on tetrathionate or sulfur were
compared (Figures 3 and 4 in paper I) and secondly, planktonic as well as
sessile cells of sulfur grown batch cultures were harvested and analyzed
(Figure 5 in paper I). Complete lists of identified, differentially expressed
proteins are supplied in the appendix of paper I. Several proteins predicted
to be involved in sulfur metabolism were differentially expressed. Proteins
encoded by both sox clusters were up-regulated during growth on
tetrathionate which was in accordance with the results from semiQ RT-PCR.
Additionally, both orthologues of Sqr were up-regulated on tetrathionate.
Several Hdr subunits were differentially expressed whereas neither Tth nor
Sor were detected in proteomics which strengthens the semiQ RT-PCR
results. These results indicated that the predicted genes were involved in
sulfur metabolism and that some of them were differentially regulated
depending on the main sulfur compound available. Furthermore, the results
from the first proteomics comparison revealed different general trends for
cells grown on tetrathionate and sulfur. During growth on tetrathionate
proteins involved in central carbon metabolism, cell division, translation,
and DNA repair were up-regulated. Up-regulation of proteins from those
functional groups might suggest that bacteria grew faster on tetrathionate
42
than on sulfur. However, since the biomass was produced in continuous
cultures with the same flow rates the growth rates were also identical.
Therefore, this trend could point to a stress response during growth on
sulfur triggering the down-regulation of central functions. Additionally, upregulation of chaperones and proteases during growth on sulfur suggested
high stress levels in this condition. Some proteins involved in carbon dioxide
fixation were also expressed in higher levels during utilization of sulfur,
however, the reason for this remains unknown.
The interpretation of the results was complicated by the fact that sulfur is
a solid substrate and bacteria can grow either attached to the substrate or in
a planktonic state. Interestingly, even though sufficient surface area was
available a subpopulation of planktonic cells remained (Arredondo et al.,
1994). Thus, a proteomic comparison of sessile and planktonic
subpopulations was carried out to study potential differences in the
subpopulations. Due to low yields of sessile cells which can be extracted from
sulfur it was necessary to grow the biomass for this experiment in large batch
cultures. The trends found in sessile and planktonic cells were not as clear
cut as in the case of the comparison between sulfur- and tetrathionate grown
cells. All subunits of Hdr were found to be up-regulated in sessile cells
whereas Sqr and proteins encoded by the sox cluster were up-regulated in
planktonic cells. Furthermore, proteins involved in amino acid biosynthesis
and central carbon metabolism were present in higher levels in sessile cells
suggesting that those are less starved than planktonic cells. Nevertheless,
chaperones, which usually indicate stress, were also higher expressed in
sessile cells. A twitching motility protein and a YVTN family beta-propeller
repeat protein up-regulated in sessile cells might be involved in attachment
to the substrate. It was suggested that the sessile cells mainly utilized the
elemental sulfur and that Hdr was important for its oxidation. In contrast,
planktonic cells might be able to use soluble sulfur compounds possibly
released from sessile cells and therefore, Sqr and Sox proteins were upregulated. However, this remains to be proven.
A model of At. caldus sulfur metabolism based on the above results is
presented in Figure 1B of paper I. The proteomics and semiQ RT-PCR results
showed clear indications that proteins encoded in both sox clusters were
involved in sulfur metabolism. The same holds true for Hdr which was
previously suggested to be involved in At. ferrooxidans sulfur oxidation
(Quatrini et al., 2009). Although there are still open questions this model
provides a good basis for further targeted investigations of implicated
enzymes.
In the meantime, Chen et al. (2012) published a refined sulfur oxidation
model for At. caldus MTH-04 based on transcriptomic analysis of the wild
type and a sor deletion mutant grown on elemental sulfur and tetrathionate.
While some aspects of the new model are essentially as proposed in paper I,
43
such as the role of the Sox system, Tth, proposed sulfide oxidation, as well as
electron transport through the quinone pool to terminal oxidases bo3, bd, or
the NADH complex, the recently presented model suggests novel oxidation
pathways for elemental sulfur. Three elemental sulfur oxidation pathways
are proposed to take place in various cell compartments: (i) a sulfur
dioxygenase (SDO) oxidizes the activated sulfur in the periplasm to yield
sulfite; (ii) Hdr oxidizes GSSH in the cytoplasm near the inner membrane to
regenerate thiol proteins (GSH); and (iii) Sor oxidizes cytoplasmic elemental
sulfur originating from periplasmic oxidation processes (e.g. produced by
SQR or the Sox system) to yield thiosulfate. The thiosulfate is proposed to be
oxidized by rhodanese using GSH as sulfur atom acceptors and yielding
GSSH which in turn is the substrate for Hdr. Furthermore, sulfur oxidation
by Sor is suggested to be coupled neither to the electron transport chain nor
to substrate level phosphorylation. Hence, in the novel model Hdr as well as
Sor are responsible for oxidation of cytoplasmic elemental sulfur preventing
the accumulation of intracellular sulfur globules. In contrast, SDO is
proposed to solely deal with sulfur of extracellular origin. The SDO enzyme
activity has been reported for acidithiobacilli but the identity of the SDO
protein remained unknown so far (Rohwerder and Sand, 2003). In the
bioinformatics analysis of paper I no putative SDO was detected.
Unfortunatelly, the data describing the cloning and characterization of the
novel SDO in At. caldus is not presented by Chen et al. (2012).
Paper II: Anaerobic sulfur metabolism coupled to dissimilatory
iron reduction in the extremophile Acidithiobacillus
ferrooxidans
The ability of At. ferrooxidans to grow anaerobically was reported many
years ago (Brock and Gustafson, 1976; Das et al., 1992; Pronk et al., 1992),
yet, the underlying mechanisms are largely unknown. Therefore, in paper II
a global transcriptomics and proteomics study of At. ferrooxidans sulfur
oxidation coupled to ferric iron reduction was conducted. Growth with the
electron donor sulfur and ferric iron as electron acceptor was compared to
growth with sulfur as electron donor and molecular oxygen as electron
acceptor. The biomass for RNA and protein extractions was grown in pH
controlled batch cultures and analysis was based on three biological
replicates. For complete lists of differentially expressed genes and proteins
see Supplemental Tables S1 and S2 of paper II1.
In aerobic as well as anaerobic condition sulfur is proposed to be oxidized
by Hdr to produce sulfite. The HdrB subunit was expressed in both
conditions but with increased levels under aerobic conditions. The same was
1 Supplemental data tables are not shown due to space limitations.
44
true for the HdrA subunit and a hypothetical protein encoded within the Hdr
gene cluster. In both conditions the sulfite generated by Hdr was proposed to
be further converted to sulfate or assimilated for growth according to the
aerobic sulfur oxidation model (Quatrini et al., 2009). Differentially
expressed genes/proteins from those pathways supported this suggestion
(Table 1 of paperII). Interestingly, the iron-binding subunit of sulfur
reductase (SreB) was uniquely expressed during anaerobic growth which
suggested the production of sulfide from reduction of sulfur by Sre under
anaerobic conditions. Furthermore, the proteins DsrE and TusA were upregulated in anaerobic conditions and thus were proposed to transfer sulfide
out of the cell. Taken together this indicated an indirect ferric reduction
pathway with the abiotic reduction of the terminal electron acceptor by the
generated sulfide. Biological production of sulfide during anaerobic growth
was evaluated by anaerobic incubation in the presence of soluble copper.
Inoculated cultures showed black precipitates, most likely copper sulfide,
and a reduced concentration of soluble copper. While in sterile controls
black precipitates were not produced. This was a strong indication of sulfide
production during anaerobic growth of At. ferrooxidans and supports the
hypothesized indirect ferric reduction pathway. Models for the aerobic and
anaerobic electron transfer and energy conservation were presented in
Figure 1 of paperII.
Previously, proteins encoded in the rus operon, TtH, or ArsH were
suggested to reduce ferric iron under anaerobic conditions (discussed in
chapter 3.3). However, the data presented here did not support the role of a
ferric reductase for any of the mentioned proteins. Although no alternative
putative ferric reductase was detected the possibility remains that ferric
reduction is mediated by an unknown ferric reductase through the bc1
complex (encoded by petII) and the cytochrome c CycA2 which were upregulated during anaerobic growth.
The data suggested that energy conservation takes place at the F0F1
ATPase. Interestingly, several of the F1 catalytic subunits were up-regulated
during aerobic growth whereas some of the F0 proton translocating subunits
were up-regulated under anaerobic conditions (Table 1 of paper II). This
indicated that more ATP was generated during aerobic growth and
generation of PMF was more important during anaerobic growth. Upregulation of the F1 subunits, i.e. increased ATP production, may be due to
an enhanced demand of ATP during aerobic growth. This in turn was most
likely due to the accelerated growth rate in the aerobic batch cultures that
were harvested after only 3-5 days compared to 2-3 weeks for anaerobic
cultures. The faster aerobic growth rate was also reflected in other trends
observed in the transcriptomics and proteomics data. Most prominently,
many enzymes of the central carbon metabolism were up-regulated during
aerobic growth (Table 1 and Figure 2 of paper II). Furthermore, proteins
45
involved in the oxidative stress response and molecular chaperone functions
were more highly expressed during aerobic growth (Table 1 of paper II)
probably reflecting both increased oxidative stress as well as increased
growth rate. In contrast, the expression patterns of several genes and
proteins involved in carbon dioxide fixation via the CBB cycle (Table 1 and
Supplemental Figure S3 of paper II) suggested an increased demand of
carbon dioxide during anaerobic growth. This was likely caused by the
intermittent supply of carbon dioxide during anaerobic cultivation.
The most striking result of the proteomics analysis was the dominance of
protein spots on the outer membrane enriched gels from the anaerobically
grown cells (Supplemental Figure S2 of paper II). Those highly abundant
proteins were identified to be OMP40 and an OMPP1/FadL/TodX family
protein. OMP40 was not expressed at significantly different levels in the two
tested conditions. The remaining seven protein spots (OMPP1/FadL/TodX
family protein) dominating the 2D gel had approximately the same MW but
various isoelectric points (pIs) which might be caused by post translational
modifications (PTMs). The fact that the expression levels of the
OMPP1/FadL/TodX family protein were so high that it dominated the spot
pattern of the 2D gel in such a way suggests that this protein had an
important function for anaerobic growth. Previously this protein has been
shown to be an OMP and to be up-regulated during aerobic growth on sulfur
compared to growth on ferrous iron (Yarzabal et al., 2003). The
OMPP1/FadL/TodX family protein was proposed to transport hydrophobic
compounds into the periplasm (van den Berg, 2010).
Paper III: Extreme zinc tolerance in acidophilic microorganisms
from the bacterial and archaeal domains
In this paper a detailed study of zinc toxicity in three acidophilic model
organisms was presented. The selected model organisms were the Gram
negative sulfur oxidizer At. caldus, the Gram positive iron oxidizer Am.
ferrooxidans, and the iron oxidizing archaeon ‘F. acidarmanus’. The basis of
this study was a bioinformatics prediction of transporters known to be
involved in neutrophile zinc resistance mechanisms. These predictions were
based on Hidden Markov Models (HMMs) and a total of seven acidophile
genomic sequences including the three model organisms were investigated.
The toxicity of zinc towards the three model organisms was assessed by
batch growth experiments while zinc speciation modeling in the media
containing either tetrathionate or ferrous iron was used to estimate the
amount of the presumed most toxic species, free ionic zinc. The biomass for
further experiments was grown in continuous culture with media containing
trace amounts of zinc and 200 mM zinc sulfate for the control and test
cultures, respectively. Inductively coupled plasma mass spectrometry (ICP46
MS) was used to measure accumulation of intracellular zinc. The levels of
transcripts of selected predicted zinc transporters were measured with
semiQ RT-PCR to check whether the predicted transporters might be
involved. The global response on the protein level was analyzed by proteomic
comparison.
The results of bioinformatics predictions using HMM were summarized in
Table 1 of paper III. The overview shows that the different acidophiles
harbor various sets of genes homologous to known zinc transporters. The
acidithiobacilli contain predicted homologues of all transporters tested
whereas T. acidophilum was the organism containing the smallest number of
predicted zinc transporters.
Further in-depth analysis was focused on the three model organisms to
elucidate their levels of zinc tolerance as well as finding clues on the
resistance mechanisms that each organism employs. To test zinc toxicity
levels batch growth experiments were carried out and substrate consumption
as well as biomass production was measured. Tolerance levels of Am.
ferrooxidans and ‘F. acidarmanus’ were comparable with a cessation of
biomass production at a zinc concentration of around 750 mM in the
medium although substrate consumption still continued at this zinc
concentration (Supplemental Files S5-S8 of paper III). In contrast, At.
caldus was inhibited at 200 mM zinc (Supplemental File S4 of paper III).
Zinc speciation modeling showed that sulfate was a suitable ligand for zinc
at low pH (Figure 1 of paper III). Acidophile media usually contain high
concentrations of sulfate that can complex zinc and mitigate its toxicity. The
model for zinc speciation in the ferrous iron containing Am. ferrooxidans
and ‘F. acidarmanus’ growth media predicted that only about 45% of total
zinc remains available as free zinc cations. Therefore, the amounts of free
zinc are much lower than it was assumed. The speciation model for the
tetrathionate containing medium of At. caldus could not be determined
conclusively as information on equilibrium constants was lacking. However,
this medium contained 50 mM less sulfate because additional sulfate was
added to the iron medium as ferrous sulfate. Hence, it is probable that more
free zinc was available in the tetrathionate medium which might partly
explain the lower tolerance levels of At. caldus. The results of the speciation
modeling highlight the importance of solution effects on the toxicity of
metals (reviewed in chapter 4 of the introduction), an aspect that has
previously not been given much attention in acidophile research. In chapter
4 of the introduction it was also pointed out that other factors, such as
competition with cations possibly including protons, can influence metal
toxicity. Such influences have not been considered with the presented
speciation modeling. Nevertheless, preliminary toxicity experiments at
various pH with At. caldus showed that zinc toxicity was lower at pH 2
compared with pH 3 suggesting that competitive effects of protons might
47
play a role in acidophile metal toxicity (Figure 6). This is an important
finding which should be kept in mind when dealing with acidophile metal
toxicity.
Figure 6. Growth of At. caldus on tetrathionate measured by generation of acidity. Preliminary data shows
the influence of solution pH on zinc toxicity. Shown are triplicate measurements with standard deviations of
cultures with starting pH of 2.0 (circles) and 3.0 (triangles) in the absence of zinc (solid symbols) and in the
presence of 25 mM zinc (open symbols). At pH 3, 25 mM zinc inhibits growth of At. caldus whereas the same
amount of zinc does not influence growth at pH 2.
The results of the transcript profiling analysis were shown in Figure 3 of
paper III and demonstrated that all targeted genes, except putative zntA of
‘F. acidarmanus’, were transcribed at similar levels regardless of the
amounts of extracellular zinc. ZntA is a primary zinc export P-type ATPase
and the putative ‘F. acidarmanus’ zntA was 1.4 fold up-regulated during
growth in high zinc conditions. This might explain how ‘F. acidarmanus’ can
avoid intracellular zinc accumulation as demonstrated by ICP-MS (Table 2 of
paper III). ‘F. acidarmanus’ was the only of the three organisms which kept
constantly low amounts of intracellular zinc. The levels of intracellular zinc
increased with fold changes of 2.6 and 5.4 (comparing highest extracellular
zinc to the control) for Am. ferrooxidans and At. caldus, respectively.
The proteomics analysis yielded a list of differentially expressed proteins
for each model organism presented in Supplemental Files S13-S16 of paper
III2. The proteomics data did not reveal specific zinc resistance mechanisms,
except a putative mechanism for At. caldus discussed below, but rather
illustrated the global response of the three model organisms to elevated zinc
levels. Supplemental File S16 of paper III shows the differentially expressed
proteins in each condition classified according to functional groups. As can
be seen, no unifying trend could be discerned among the three acidophiles.
Interestingly, no chaperones pointing to a stress response were detected as
2 Supplemental data tables are not shown due to space limitations.
48
differentially expressed. In all tested organisms several enzymes involved in
central carbon metabolism were differentially expressed. Yet, those enzymes
showed various expression patterns suggesting that carbon flow was
individually adjusted in response to high extracellular zinc. Noteworthy are
two proteins involved in oxidative stress response: alkyl hydroperoxidase
reductase up-regulated in At. caldus on high zinc, and OsmC family protein
down-regulated in Am. ferrooxidans on high zinc (Supplemental File S17 of
paper III). This suggests different roles of zinc with respect to oxidative
stress in the two acidophiles. Finally, one subunit of a phosphate ABC
transporter, PstS was up-regulated during growth on high zinc in At. caldus
and it was hypothesized that a polyP-based resistance mechanism was in
operation similar to the copper resistance mechanism described for
acidophiles (Orell et al., 2010). This resistance mechanism is based on
sequestration of metal ions by intracellular polyP, cleavage of polyP, and
export of phosphate-bound metal ions by phosphate transporters. However,
whether At. caldus employs such a resistance mechanism in case of zinc
could not be shown in the present study and requires further investigation.
Paper IV: Response of Acidithiobacillus caldus towards
suboptimal pH conditions
In order to study which proteins were important for pH homeostasis in At.
caldus a proteomics analysis of cells grown at three different extracellular
pH values was carried out. At. caldus was grown under tightly controlled
conditions in continuous culture with a pH control system keeping constant
pH at either pH 1.1 (below optimal), optimal pH 2.5 (optimal), or pH 4.0
(above optimal). To further elucidate changes in the cytoplasmic membrane,
profiles of membrane fatty acid methyl esters (FAMEs) were analyzed by GCMS.
The proteomics analysis revealed major adjustments of the proteome in
response to extracellular pH. A complete list of identified, differentially
expressed proteins was presented in Supplemental Table 1 of paper IV3. A
summary of the differentially expressed proteins discussed in more detail
was shown in Table 1 of paper IV. Various transcription factors showed
specific expression patterns upon changes of extracellular pH (examples
shown in Figure 2 of paper IV) suggesting that a tight transcriptional control
was important. One of the transcription factors that showed highest
expression in pH 1.1 was PspA, a protein initially studied in E. coli phage
shock response. More recently a direct role of PspA in the maintenance of
PMF in neutrophiles was reported (Kobayashi et al., 2007) as well as a
connection between Psp response and acid stress (Jovanovic et al., 2006).
3 Supplemental data files are not shown due to space limitations.
49
Apart from transcriptional control there were also indications of specific
PTM in response to extracellular pH (Figure 1 paper IV). Some PTMs like
serine, threonine, and tyrosine phosphorylation are detectable in 2D-PAGE
gels as they are stable under the experimental conditions and detectably alter
the pI of the protein. Thus, proteins modified in such a way are visible as
discrete spots with similar MW but shifted pI compared to the unmodified
protein. Examples of protein spot patterns that could be explained with this
phenomenon were found for the phosphate selective porin O and P (Figure 1
paper IV) and Sqr (Figure 3 paper IV). Clearly, this phenomenon requires
further investigations to prove whether protein phosphorylation is its cause
and to elucidate the biological function of this modification.
Another obvious trend was up-regulation in low pH of enzymes involved
in sulfur metabolism such as Sqr, Hdr, or Sox proteins (Figure 3 of paper
IV). All of these proteins showed elevated levels at pH 1.1 which suggested
either that cells needed more energy to grow at low pH and therefore, they
enhance the oxidation of their energy substrate or that sulfur oxidation
enzymes are specifically up-regulated to allow increased outward proton
pumping by primary electron transport proton pumps. The latter
explanation would comprise a specific pH homeostasis mechanism in At.
caldus and supporting evidence for the connection between sulfur oxidation
and PMF was previously reported (Dopson et al., 2002).
Other differentially expressed proteins were of interest as they had been
shown to be involved in acid stress responses of neutrophiles (Figure 4 of
paper IV). These included a toluene tolerance protein up-regulated at more
basic pH, spermidine synthase detected in two protein spots with different
expression patterns, and glutamate decarboxylase which was a unique
protein spot at pH 1.1. The toluene tolerance protein was encoded together
with other proteins putatively involved in hopanoid biosynthesis. Hopanoids
are bacterial sterol analogues which might play a role in membrane
adjustments in response to environmental conditions. Spermidine and other
polyamines have various roles in bacteria (Wortham et al., 2007), however,
reports have shown their involvement in acid stress response (Samartzidou
et al., 2003). Although the expression patterns in the present study were not
obviously related to an acid stress response the differential expression of
spermidine synthase might point to a pH homeostatic role of spermidine in
At. caldus. However, the most interesting protein in this context was
glutamate decarboxylase which is part of the most important acid stress
resistance system in E. coli whereby protons are intracellularly consumed by
decarboxylation of glutamate. Whether a similar system operates in At.
caldus needs further investigation, however, it is intriguing that glutamate
decarboxylase was expressed at high levels only in the low pH 1.1 (Figure 4 of
paper IV).
50
FAME analysis clearly showed a modulation of the cytoplasmic membrane
in response to proton concentration with the largest changes at lowest pH
(Figure 5 of paper IV). Similar to the low pH response of At. ferrooxidans
(Mykytczuk et al., 2010) FAMEs C16:0 were increased and C18:1 were
decreased in At. caldus. Such changes likely brought about a more rigid
membrane which would be more efficient in shielding protons from entering
the cell. In contrast to At. ferrooxidans (Mykytczuk et al., 2010) and E. coli
(Chang and Cronan, 1999) the At. caldus cyclo-C19:0 content was decreased
upon growth in low pH. Interestingly, the content of cyclo-C19:0 was highest
during growth at optimal pH and reduced to about the same levels at pH 1.1
and 4.o. The biological meaning of this phenomenon remains unknown.
The results of this paper showed very interesting trends in response to
suboptimal extracellular pH. So far it has not been shown that neutrophile
acid resistance mechanisms could be in operation in acidophiles during
growth in very low pH. This was an interesting finding that needs further
verification. Therefore, the presented paper supplies a good basis for further
studies on pH homeostasis in At. caldus.
51
Conclusions
Proteomics can yield insights into general responses as well as giving clues
regarding specific mechanisms for adaptation to life in extreme
environments.
Sulfur oxidation pathways in acidophiles were diverse, even within
acidithiobacilli. At. caldus used the Sox system as well as Sor which were not
present in At. ferrooxidans. However, both acidithiobacilli were proposed to
oxidize sulfur via Hdr.
Anaerobic sulfur oxidation in At. ferrooxidans was, at least in part, indirectly
coupled to ferric iron reduction via sulfide generation.
Speciation of metals and other chemical influences were of great importance
for the toxicity of zinc in acidophiles. At. caldus, Am. ferrooxidans, and ‘F.
acidarmanus’ showed distinct responses to elevated zinc levels.
Solution pH, especially values below growth optimum, greatly influenced the
At. caldus protein expression pattern and FAME profiles. Active adjustments
of FAME composition likely yielded a more rigid membrane at lower pH.
Moreover, indications for the use of acid resistance mechanisms similar to
neutrophilic systems were found.
52
Future Directions
Research on acidophiles is of great importance for human society as insight
into geomicrobiological and geochemical processes will lead to benefits for
society such as the control of detrimantal effect caused by microorganisms
and the exploitation of acidophiles for industrial processes. Undoubtedly,
acidophile research has taken a leap forward with entering the genomic and
post-genomic era. However, we are just beginning to understand the
fundamental processes associated with an acidophilic lifestyle and many
open questions remain to be answered. As pinpointed within this thesis the
application of ‘omics’ techniques has advanced our knowledge concerning
many aspects of acidophile microbiology and has created a great deal of
hypotheses that await scrutinization. Especially, the recently reported
successes with genetic manipulation of acidophilic microorganisms,
specifically acidithiobacilli, open up new technical possibilities. This will
further spur the research in the field and help to solve important questions.
Furthermore, systems biology approaches start to emerge within the field of
acidophile research which will help to integrate knowledge and provide
deeper comprehension than regarding isolated aspects. Yet, a note of caution
is appropriate here as systems biology not only offers great opportunities but
also bears pitfalls such as computational limitations in dealing with
enormous amounts of data. Hence, research has to develop in the field of
bioinformatics as well in order to circumvent technical problems.
Some findings of this thesis are of particular interest and should be
further studied to elucidate the underlying processes. The model for At.
caldus sulfur metabolism presented here was recently further refined but
still the sulfide oxidation in the cytoplasm remains an unsolved aspect.
Furthermore, the identity and characteristics of the GSSH oxidizing enzyme
Sdo, the main novelty of the recent model, remain elusive. This is an
extremely important aspect of the sulfur oxidation pathway that has also
implications for At. ferrooxidans sulfur oxidation since the presence of Sdo
in At. ferrooxidans was hypothesized earlier. The recent model proposes a
compartementalization of sulfur oxidation in At caldus and suggests the
prevention of sulfur globule formation by cytoplasmic sulfur oxidation
carried out by Sor. Clear evidence for the lack of intracellular sulfur globules
in At. caldus is missing. However, the presence of sulfur globules would also
affect the hypothesized metal resistance mechanism based on polyP
sequestration and export suggested in this thesis. Therefore, proofing the
absence or presence of intracellular sulfur globules in At. caldus would add
another piece to the puzzle.
Very interesting was the discovery that At. ferrooxidans anaerobic sulfur
oxidation is, at least in part, indirectly coupled to ferric reduction through
53
biological production of sulfide. As a matter of fact At. ferrooxidans is able to
utilize hydrogen oxidation coupled to ferric reduction where sulfide
generation would not occur and therefore, alternative pathways for ferric
reduction are necessary. Hence, the clarification of ferric reduction by At.
ferrooxidans is still not complete. Additionally, the proteomics analysis
provided an exciting clue on the importance of an OMPP1/FadL/TodX
family protein for anaerobic growth. Creating a deletion mutant for this
protein might help to reveal its biological function.
Additionally, the proteomics study of At. caldus pH homeostasis provided
several interesting hypotheses that should be tested in depth. The most
exciting finding was the indication that At. caldus might utilize a
decarboxylase system during acid stress as reported for neutrophilic acid
stress systems.
In all the proteomics studies presented in this thesis were indications that
protein isoforms played a biological role for the characteristics under
observation. Protein isoforms with similar MW but significantly shifted pIs
were probably due to PTMs. This is an important observation and
investigation of those protein isoforms with specialized proteomics
techniques will help to understand cellular processes on a molecular scale.
A significant finding of this thesis was that zinc toxicity in acidophiles was
largely influenced by speciation and chemical effects occurring in solution,
such as effects due to pH. So far, such aspects were to a large extent not
considered for metal toxicity in acidophiles. It will be beneficial to further
investigate metal toxicity in dependence of solution chemistry and to bear
those influences in mind when making comparisons between metal toxicity
of various species. This finding also underscores the importance of
interdisciplinary research especially in the field of acidophile research which
is heavily interconnected with other fields such as mineralogy, geology, and
geochemistry.
54
Acknowledgements
For scientific guidance, advice, and discussions, I am very grateful to
Mark Dopson
Joanna Potrykus
Maria Liljeqvist
Siv Sääf
Olena Rzhepishevska
Wolfgang Schröder
Marc Röhm
Previous members of group MDo
All collaborators who contributed to the manuscripts
Administration of Molecular Biology, Media, and Johnny Stenman for
support in the daily life of a PhD student
For everyone who left their footprints in my life. Thank you for everything!
Mohammad Khoshkhoo, Renaud Lazer, Maria Liljeqvist, Junfa Liu and his
family, Hande Öztokatli, Joanna Potrykus, Congting Qi, Marc Röhm, Olena
Rzhepishevska and her family, Martin Schmid and his family, Meir Sussman,
Hao Xu
Von ganzem Herzen danke ich meinen Freunden in Deutschland, Markus
Wagner und Christine Fürniß – was würde ich ohne Euch tun?
Und schließlich danke ich meiner Familie, dem Wertvollsten, das ich besitze,
Matthias, Brigitte und Fritz Mangold sowie Oliver, Ingrid und Günter
Schwahn.
55
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