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Regulation of nitrogen fixation in Rhodospirillum rubrum Tiago Toscano Selão

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Regulation of nitrogen fixation in Rhodospirillum rubrum Tiago Toscano Selão
Regulation of nitrogen fixation in
Rhodospirillum rubrum
Through proteomics and beyond
Tiago Toscano Selão
©Tiago Toscano Selão, Stockholm 2010
ISBN 978-91-7447-125-0, pp 1 – 71
Printed in Sweden by US-AB, Stockholm 2010
Distributor: Department of Biochemistry and Biophysics
Cover image: Three-dimensional rendering of the Gaussian peaks for a 2D-PAGE gel, generated
using PDQuest 7.3.0.
Dedicado aos meus pais e
à memória dos meus avós.
Publication list
The work presented on this thesis is based on the following
publications, referred to in the text by the corresponding Roman
numerals:
I – Selão, T. T., Nordlund, S. and Norén, A. ”Comparative proteomic
studies in Rhodospirillum rubrum grown under different nitrogen
conditions”, J Prot Res, 2008, 7: p. 3267-75
II – Teixeira, P. F.*, Selão, T. T.*, Henriksson, V., Wang, H., Norén,
A. and Nordlund, S. ”Diazotrophic growth of Rhodospirillum rubrum
with 2-oxoglutarate as sole carbon source affects the regulation of
nitrogen metabolism as well as the soluble proteome”, Res. Microb.,
2010, in press
III – Selão, T. T., Branca, R., Lehtiö, J., Chae, P. S., Gellman, S. H.,
Rasmussen, S., Nordlund, S. and Norén, A. ”Identification of the
chromatophore membrane complexes formed under different nitrogen
conditions in Rhodospirillum rubrum”, submitted
IV – Selão, T. T.*, Teixeira, P. F.* and Nordlund, S. ”The activity of
dinitrogenase reductase ADP-ribosyltransferase of Rhodospirillum
rubrum is controlled through reversible complex formation with the
PII protein GlnB”, manuscript
V – Selão, T. T., Edgren, T., Wang, H., Norén, A. and Nordlund, S.
”The effect of pyruvate on the metabolic regulation of nitrogenase
activity in Rhodospirillum rubrum with darkness as switch-off
effector”, submitted
* Authors contributed equally
Additional publications
Vintila, S., Selão, T. T., Norén, A., Bergman, B., El-Shehawy, R.,
“Characterization of nifH gene expression, modification and
rearrangement in Nodularia spumigena strain AV1”, submitted
Contents
I. Introduction....................................................................... 9
1. Bacteria, the largest population of Earth ...................................... 9
2. One of the elements of life - nitrogen........................................... 9
3. Nitrogen fixation and assimilation ............................................. 11
3.1. The diazotrophs .......................................................................................11
3.2. Nitrogenase and nitrogen fixation............................................................13
3.3. Nitrogen assimilation...............................................................................17
3.4. Regulation of nitrogen fixation and assimilation .....................................19
4. An overview of the field of proteomics...................................... 34
4.1. Gel-based proteomics ..............................................................................34
4.2. Gel-free proteomics .................................................................................37
4.3. Mass spectrometry ...................................................................................37
II. Adaptation to different nitrogen conditions .................. 40
1. The interconnection between nitrogen fixation and carbon
fixation pathways ........................................................................... 40
2. Keeping the (redox) balance....................................................... 42
III. Post-translational regulation of nitrogenase activity –
guarding the guards ............................................................ 44
1. Regulation of ammonium switch-off ......................................... 44
2. Regulation of energy switch-off................................................. 46
IV. Future prospects ........................................................... 50
V. Concluding remarks....................................................... 51
VI. Sumário em português ................................................. 53
VII. Acknowledgements..................................................... 55
VIII. References ................................................................. 58
Abbreviations
AAA+
Arc
FNR
GAF
Km
SDS-PAGE
σ
ATPase associated with diverse cellular activities
Aerobic respiratory control
Fumarate and nitrate reduction regulator
cGMP phosphodyesterase; adenylyl cyclase; FhlA
Michaelis-Menten constant
Sodium dodecylsulphate – polyacrylamide gel
electrophoresis
Sigma subunit of the RNA polymerase
“Cantando espalharei por toda parte,
Se a tanto me ajudar o engenho e arte. [...]
E vós, Tágides minhas, pois criado
Tendes em mi um novo engenho ardente
Se sempre, em verso, humilde, celebrando
Foi de mi vosso rio alegremente,
Dai-me agora um som alto e sublimado,
Um estilo grandíloco e corrente
Por que de vossas águas Febo ordene
Que não tenham enveja às de Hipocrene.
Dai-me hũa fúria grande e sonorosa,
E não de agreste avena ou frauta ruda,
Mas de tuba canora e belicosa,
Que o peito acende e a cor ao gesto muda.”
Luís Vaz de Camões, in “Os Lusíadas”, Canto I
8
I. Introduction
1. Bacteria, the largest population of Earth
The multitude of forms of life present all around us is estimated to
have arisen from a single common ancestor over 3,5 billion years ago
1
. Of all the different organisms that science has now described,
bacteria are undoubtedly the most widely spread and adaptable of all.
They have been found in all different kinds of habitats on Earth, from
deep within the planet’s crust to the surface of the seas, living within
mammalian intestines or in acid mine drainage. Deinococcus
radiodurans is perhaps the best example of the extreme hardiness of
bacteria, being able to survive exposure to cold, large pH variations,
dehydration and, for what it is most known, high doses of ionizing
radiation 2. The extreme adaptability of bacteria has allowed them to
become ubiquitous on our planet, to the extent that they are estimated
to be the largest contributors to the world’s biomass 3. It was even
recently suggested that complex animals, including humans, should
actually be seen as “super-organisms”, in view of the fact that they
host about ten times as many bacterial cells as their own and that these
bacteria actually interact and affect the animals’ own metabolism 4.
2. One of the elements of life - nitrogen
In 1772 Daniel Rutherford, a Scottish chemist and physician,
described the presence of a gas or “air”, as it was then called, in the
atmosphere. By removing what Joseph Black had named in 1754 as
“fixed air” (carbon dioxide) from the atmosphere, generated by
burning coal, he isolated what he entitled “noxious air”. It was later
named “azote” (“lifeless”) by Antoine Lavoisier 5 as animals would
suffocate in it and even the flame of a candle would quickly die. The
9
work of Lavoisier and, almost simultaneously of Joseph Priestley and
Carl Wilhelm Scheele made clear that “azote”, or nitrogen, was indeed
an elementary constituent of not only air but also of many other
substances.
The name “nitrogen”, most commonly used in the English language,
derives from the Greek word “νιτρον” (“nitron”), nitre or saltpetre
(potassium nitrate), a compound used in the manufacture of
gunpowder and soap and also used as a fertilizer. The root of the word
denotes the fact that even though it was not until the 18th century that
nitrogen was recognized as an element, manufactured substances
containing it had been in use for centuries.
Nitrogen is one of the most common elements in biomolecules and
can very frequently become a growth limiting factor for living beings,
due to the fact that it is not always available in a biologically usable
form. The triple bond in molecular nitrogen (N2) is the strongest
among naturally occurring substances (bond dissociation enthalpy of
946 kJ.mol-1) 6, giving rise to the unreactiveness of atmospheric
nitrogen. This high energy threshold makes atmospheric dinitrogen
biologically unavailable to most organisms. However, a group of
prokaryotes produces an enzyme complex, nitrogenase, which
catalyzes reduction of atmospheric dinitrogen to ammonium, at room
temperature and pressure, thus replenishing the biological nitrogen
pool (Figure 1). This amazing feat of evolution becomes even more
astonishing if we bear in mind that ammonium production at an
industrial scale by the Haber-Bosch process requires a very high
energy input, with temperatures in excess of 300 ºC, pressures of 1520 MPa and specific catalysts.
Figure 1 – The global nitrogen cycle
10
3. Nitrogen fixation and assimilation
3.1. The diazotrophs
Diazotrophic bacteria are responsible for an important step in the
global nitrogen cycle, the reduction of atmospheric dinitrogen to
ammonium. The capacity to perform this reaction is exclusive of
prokaryotic organisms. Several symbiotic relations between plants and
diazotrophs are also well established, thus allowing eukaryotic
organisms to directly harness the power of nitrogen reduction.
Symbiotic diazotrophs interact with plants using three different
strategies. Endosymbiotic diazotrophs are perhaps the most widely
known type of symbiotic nitrogen fixing bacteria, infecting the roots
of plants and establishing an elaborate type of intracellular symbiosis,
in which extensive signalling pathways exist between host and
symbiont. A well-studied example is the relation between
Rhizobiaceae and leguminous plants, such as pea or soybean, with the
infection by these bacteria inducing formation of root nodules in the
plant and resulting in morphological and metabolic changes in the
bacteria themselves, ultimately leading to their differentiation into
nitrogen fixing “organelles” named "bacteroids" 7. These symbiotic
organisms are the basis for the agricultural practice of crop rotation,
used by farmers around the world for generations to increase yields.
Other symbiotic diazotrophs, termed endophytic, establish
themselves within the vascular tissue of the host plants, as is the case
for Gluconoacetobacter diazotrophicus and Herbaspirillum
seropedicae and their host, sugar cane 8, 9 or Azoarcus sp. and its
symbiosis with rice and Kallar grass 10. Finally, some of these bacteria
can interact in a rather non-invasive and to some extent less elaborate
form with their hosts, by colonizing the surface or specific cavities of
plants. These associative symbionts are exemplified by the relations
established between Azospirillum spp. and different kinds of grasses
11
.
On the other hand, most nitrogen fixing bacteria can be found in a
free-living form. Already in 1893 Winogradsky demonstrated that
Clostridium pasteurianum could use atmospheric dinitrogen 12 and,
since then, many other organisms were shown to be able to perform
nitrogen fixation. Some of the most widely studied examples include
the γ-proteobacteria Klebsiella pneumoniae and Azotobacter
11
vinelandii, with the first crystal structure of the nitrogenase complex
obtained using the proteins from the latter organism 13.
Free-living diazotrophic organisms are found in a variety of
environments and are usually classified as aerobic, anaerobic or
photosynthetic. This last group is of special interest, not only for the
connection between these two very important processes
(photosynthesis and nitrogen fixation) but also for some of the
particular aspects regarding regulation of nitrogen fixation that were
first elucidated in purple non-sulphur phototropic bacteria of the genus
Rhodospirillaceae. The work described in this thesis focuses on
nitrogen fixation regulation in Rhodospirillum rubrum, a member of
the Rhodospirillaceae family.
3.1.1. The main character, Rhodospirillum rubrum
In 1887 Erwin von Esmarch described a micro organism that he had
found living in a sample of Berlin’s tap water in which a dead mouse
had been incubated. He could successfully isolate this micro organism
and observe its “beautiful wine-red colour”, the spiral shape of the
cells and the fact that stab cultures would lose their characteristic
pigmentation in the part exposed to oxygen 14. He named this
organism, due to its colour and shape, “Spirillum rubrum”. Later on,
the same organism was shown to live in freshwater sediments
(perhaps from there contaminating the water sample used by Esmarch)
all around the world and to be a purple non-sulphur photosynthetic
bacterium by Hans Molich, who then renamed it “Rhodospirillum
rubrum” 14.
R. rubrum is an α-proteobacterium, able to grow on a very wide
selection of conditions and substrates. It is a Gram-negative bacterium
and its inner membrane forms invaginations that upon cell breakage
result mostly in inside-out vesicles. These vesicles, called
chromatophores, were found ideal for bioenergetics studies, as they
contain the photosynthetic apparatus and the ATP synthase complex,
among other components 15-17. R. rubrum was the model organism
used to study, among other phenomena, the connection between cell
illumination and oxidation of cytochromes 18, as well as
photophosphorylation and light-induced redox reactions, using
chromatophore preparations 19-21.
In 1949 Kamen and Gest described the light-dependent hydrogen
production and nitrogen fixation by R. rubrum 22, 23. It was the first
12
time such activity was demonstrated in photosynthetic bacteria and
later it was also in R. rubrum that the metabolic regulation of
nitrogenase by ADP-ribosylation was demonstrated for the first time
24-26
.
R. rubrum is a very versatile organism and can grow both aerobically
in the dark and anaerobically in the light, using several carbon and
nitrogen sources 27. Its genome is publicly available and annotated at
the US Department of Energy Joint Genome Institute
(http://www.jgi.doe.gov), a tool that greatly facilitates genomic and
proteomic studies.
3.2. Nitrogenase and nitrogen fixation
The enzyme complex responsible for reduction of dinitrogen is, as
mentioned, nitrogenase. Even though the presence of nitrogenase had
already been known at the time it was not until 1960 that Carnahan
and collaborators succeeded in preparing cell-free extracts with
nitrogenase activity from C. pasteurianum 28. They also described that
its activity would be lost within 30 seconds when extracts were
exposed to oxygen. This extreme sensitivity to oxygen is due to
particular details of the structure of the complex, as we shall discuss
further on, and hints at the possibility that this enzyme system could
have evolved prior to the existence of an oxidizing atmosphere 29.
Three main types of nitrogenase have been described so far. The
most common and most widely studied form is the molybdenumcontaining type 30, 31. A second type of enzyme is usually referred to
as “alternative nitrogenase”, as it contains either vanadium or iron, in
alternative to molybdenum 32. The different nitrogenases share many
characteristics and some organisms have the capacity to express two
(as R. rubrum) or even three types of nitrogenase, although not
simultaneously 33, 34. The alternative nitrogenases are usually only
expressed when Mo concentrations in the environment are limiting
and it has been suggested that these systems could be primitive
lineages of the nitrogenase enzyme that have been kept throughout
evolution 35. A third kind of nitrogenase has been found only in
Streptomyces thermoautotrophicus. This is a very dissimilar type of
nitrogenase, depending on superoxide oxidation for activity 36 and
several of the details regarding its reaction mechanism still await
further study.
13
The molybdenum-containing nitrogenase (or simply “nitrogenase”,
as it shall henceforth be referred to) is a complex of two
metalloproteins, dinitrogenase reductase or iron protein (Fe protein)
and dinitrogenase or molybdenum-iron protein (MoFe protein) 37, 38.
The atomic structure of the nitrogenase complex (from A. vinelandii)
is shown in Figure 2A. As can be seen, dinitrogenase is an α2β2
heterotetramer, with two NifD and two NifK subunits whereas
dinitrogenase reductase is a homodimer of the NifH protein 37, 39-41.
Nitrogen reduction takes place deep within the NifD subunit of
dinitrogenase, where the iron-molybdenum cofactor (FeMo-co)
resides 42, 43. At the interface between NifD and NifK subunits in the
αβ dimer lies an [8Fe:7S] cluster, the P-cluster 43, 44. The NifH dimer
possesses a [4Fe:4S] cluster, at the interface between monomers, as
well as MgATP binding and hydrolysis sites 45. It is through this
metallocluster that electrons are transferred from the physiological
electron donors to the P-cluster in dinitrogenase and from this to the
FeMo-co and dinitrogen (Figure 2A). All of these metalloclusters are
extremely sensitive to oxidation, thus explaining the sensitivity of
nitrogenase to oxygen 46.
The synthesis, maturation and insertion of the FeMo-co into the core
of dinitrogenase require an elaborate network of several gene
products, part of the nif (nitrogen fixation) gene cluster and, in a series
of recently published experiments, this process could be reconstituted
in vitro 41, 47.
As mentioned above, electrons are transferred from the iron-sulphur
cluster within the Fe protein to the P-cluster and FeMo-co in a NifDK
dimer. The Fe protein physically docks to the MoFe protein and, after
electron transfer, hydrolysis of the MgATP molecule bound to the Fe
protein will result in dissociation of the nitrogenase complex. Each
ATP hydrolysis cycle (2 ATP per cycle) results in one electron
transferred to the P-cluster (Figure 2B), making nitrogen reduction a
highly demanding reaction 48-51. A minimum of eight electrons and,
correspondingly, 16 ATP molecules are required for reduction of one
dinitrogen molecule to ammonium. As part of the nitrogen reduction
reaction, protons are always reduced in at least a 2:1 molar ratio to N2,
even if the latter is present at very high concentrations 52, 53 (Equation
1).
N2 + 8 H+ + 8 e- + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi
(Equation 1)
14
A
B
Figure 2 – Three-dimensional structure and turnover cycle of the 2:1 nitrogenase
complex, stabilized by ADP-AIF4–. A – Atomic structure generated using the
coordinates for the A. vinelandii enzyme (PDBID: 1N2C) and PyMol 1.0. NifDK
subunits are coloured in shades of blue and NifH in green. The bound ATP and
metal centers are only highlighted in one half of the complex, for simplicity. B –
Nitrogenase turnover cycle. Nitrogenase subunits are coloured as in Figure 2A.
Colour of the [4Fe-4S] cluster of NifH denotes oxidation state - red, reduced; green,
oxidised. Adapted from 54. The components are not to scale.
Nitrogenase can also catalyze the reduction of a variety of other
substrates including acetylene 53, 55. The acetylene-reducing capacity
of this complex plays a major role in nitrogen fixation research as the
product of reduction, ethylene, can be measured using gas
chromatography 53. This method is easier to use, faster, less expensive,
and more adaptable to field work than the previously used manometric
methodologies and is still the method of choice employed in nitrogen
fixation studies in laboratories throughout the world.
15
3.2.1. Electron transport to nitrogenase in R. rubrum
The electron transport chain to nitrogenase is in many diazotrophs
poorly characterized and can have different unique aspects depending
on the organism. Nevertheless, all nitrogenase enzymes require low
potential reducing equivalents. Work in our laboratory in recent years
has shed some light on the electron transport to nitrogenase in R.
rubrum. Two pathways were found to be operating in R. rubrum
nitrogen fixing cells, with the major pathway operating under
photoheterotrophic conditions and using the FixABCX proteins 56, 57.
The Fix system is similar to the electron transfer flavoprotein (ETF)
and electron transfer flavoprotein ubiquinone-oxidoreductase (ETFQO) involved in electron transfer reactions in both mitochondria and
bacteria 58, even though in R. rubrum both fixABCX and “typical”
ETF/ETF-QO genes can be found, just as in other diazotrophic
bacteria, such as Bradyrhizobium japonicum 59.
R. rubrum FixC was shown to associate to the membrane and to bind
FAD 56. FixX is a ferredoxin-like protein, with an iron-sulphur cluster
binding motif, showing similarity to ETF-QO. R. rubrum FixA and
FixB are similar to ETF and were shown to have NAD(P)H
dehydrogenase activity and bind one FAD molecule per dimer 60. It
was proposed that FixAB could be reduced by NADH and the
electrons then transferred to the FAD moiety in FixC. FixC would
deliver one of the electrons up-gradient to the iron-sulphur cluster in
FixX and another electron down-gradient to ubiquinone in the
membrane, generating reduced ubiquinol that would be reoxidized by
reversed electron flow, generating NADH 60. This reversed electron
flow would be supported by the proton motive force generated by
photosynthetic activity (see Figure 3) and some data exists supporting
the importance of an energized chromatophore membrane for the
process of nitrogen reduction in R. rubrum 61 as well as in other
diazotrophic bacteria 62, 63. A similar process has also been proposed
to occur in photoheterotrophically grown Rb. capsulatus, as a means
to maintain redox balance in the cell 64.
The second electron pathway to nitrogenase in R. rubrum includes
NifJ, a pyruvate:ferredoxin oxidoreductase, which can directly link the
oxidation of pyruvate to reduction of ferredoxin 65, 66. This pathway
was shown not to be required for nitrogen fixation under
photoheterotrophic conditions 57, although it could play an important
role under anaerobic conditions in the dark. In both cases the major
16
direct electron donor to dinitrogenase reductase was shown to be the
ferredoxin FdN 67.
Figure 3- Proposed mechanism for electron transfer to nitrogenase through the
FixABCX system, using reversed electron flow. The proton gradient generated by
photosynthesis is used by NADH dehydrogenase (I) to reoxidise the quinone pool,
generating NADH. Succinate dehydrogenase (II) activity and electron transfer from
FixC reduce the quinone pool. The different components are not to scale.
3.3. Nitrogen assimilation
Ammonium, once produced by the nitrogenase reaction or obtained
from the surrounding environment, is assimilated in the form of amino
acids. There are two major pathways for nitrogen assimilation, one
through glutamine synthetase (GS) and glutamate:2-oxoglutarate
aminotransferase (GOGAT), also named the GS/GOGAT cycle 68, and
another using glutamate dehydrogenase (GDH). In both of these cases,
the end-products are glutamate and glutamine. We shall now examine
these two pathways in more detail.
3.3.1. The GS/GOGAT cycle
Glutamine synthetase is a key enzyme in bacterial metabolism as it
provides the only reaction for glutamine biosynthesis. Due to its vital
role, GS deletions will result in glutamine auxotrophy 69.
The GS/GOGAT cycle is the preferred means for nitrogen
assimilation in energetically favourable, nitrogen-poor conditions 70,
17
71
. In it, ammonium is condensed with glutamate to yield glutamine, a
reaction that depends on hydrolysis of ATP 72 (Equation 2). Following
glutamine synthesis, GOGAT will use it to transaminate one molecule
of 2-oxoglutarate (2OG) to glutamate, thus completing the cycle and
releasing one molecule of glutamate for metabolic purposes 69 – see
Equation 3.
NH4+ + glutamate + ATP glutamine + ADP + Pi
(Equation 2)
Glutamine + 2OG + NADPH 2 glutamate + NADP+
(Equation 3)
The net result of the action of these two enzymes is thus the
biosynthesis of a biological amino group donor (glutamate), at the
expense of ATP and reducing power. This pathway is preferred in
conditions of nitrogen deficiency over the glutamate dehydrogenase
pathway owing to the low Km for ammonium (0.1 mM) and glutamine
(0.25 mM) of GS and GOGAT, even if it requires an “extra” input of
energy in the form of ATP 72, 73. Both GS and GOGAT have been
purified from R. rubrum and partially characterized 74, 75. GS, as we
shall discuss later on, is tightly regulated, a fact that can be related to
the energetic demands of the overall reaction.
3.3.2. Glutamate dehydrogenase
The second pathway for nitrogen assimilation utilizes glutamate
dehydrogenase (GDH). This enzyme, unlike the GS/GOGAT cycle,
does not require ATP hydrolysis, condensing ammonium with the
carbon skeleton of 2OG, directly producing glutamate, at the expense
of NAD(P)H 69 – Equation 4.
NH4+ + 2OG + NAD(P)H glutamate + NAD(P)+
(Equation 4)
Escherichia coli GDH has a higher Km for ammonium than GS and is
mostly used in environments where ammonium supplies are plentiful.
Also, as it does not require ATP hydrolysis, GDH activity is usually
correlated with lower energy conditions 70, 71.
18
3.4. Regulation of nitrogen fixation and assimilation
As can be easily deduced from all the equations presented above and
from the complexity of the synthesis, maturation and insertion of the
FeMo-co into nitrogenase, all of these processes require a very large
input of energy and cellular resources. We shall now scrutinize how
the processes of nitrogen fixation and assimilation are regulated, with
special focus on the case of R. rubrum. Given that the PII proteins
play a major role in the regulation of both processes, we begin by
turning our attention to them.
3.4.1. The role of the PII proteins in nitrogen fixation and
assimilation
It was through the pioneering work of Bennett Shapiro that the PII
proteins were first discovered. While trying to isolate the GSmodifying enzyme, Shapiro separated two protein constituents as gel
filtration peaks, which he named PI and PII 76. His work established
that adenylylation of GS could only be obtained if the two fractions
(PI and PII) were present, along with ATP and 2OG. It took more than
ten years before the PII fraction could be identified in Klebsiella
aerogenes fractions as the protein GlnB 77. Since then, the importance
of PII proteins and their widespread presence throughout all domains
of life have been well established 78.
Presently, the designation “PII proteins” encompasses what is known
as a “superfamily” of proteins. These are usually small proteins, of
roughly 12-13 kDa, forming (homo)trimeric complexes. PII proteins
have a few loop regions, one of which, a protruding, flexible loop
named T-loop is of key importance to signalling and protein-protein
interactions 79, 80.
PII proteins are usually divided into 3 subgroups, each named after
an archetype protein, regarding their different functions in cellular
processes, gene organization and protein sequence. These are named
GlnB-like, GlnK-like or NifI-like 78. Proteins of the first subgroup,
including all GlnB-like proteins and named after the homonymous
protein of E. coli, are found in monocistronic operons or cotranscribed with either glnA (the gene encoding GS) or nadE
(encoding an ammonium-dependent NAD synthetase) 78.
The genes encoding the proteins in the second subgroup, named after
the second PII paralog found in E. coli, GlnK 81, are usually found in
an operon with the gene encoding the ammonium transport protein
19
AmtB 82. However, it has become clear in recent years that this is not
always the case, as in Azospirillum brasilense, where the glnZ gene
encodes a GlnK-like protein, despite the fact that it is not linked to an
amtB gene. These proteins are then classified as GlnK-like based also
on sequence similarity 78.
While these two first groups can sometimes have overlapping
functions in the cell, the third group (NifI-like) is rather distinct from
the first two, containing the PII-like proteins found in association with
the nifH gene in Euryarchaeota, clostridia, chlorobia and δproteobacterial diazotrophs 83. These usually occur in pairs (nifI1 and
nifI2), also known as nifH-proximal and nifH-distal genes. The
functions of NifI-like proteins are also distinct from that of other PII
proteins, being involved in post-translational regulation of nitrogenase
activity by direct interaction with dinitrogenase (see section 3.4.3) 84.
PII proteins are signal integrator molecules known for their roles in
signalling and regulation of metabolic processes, through proteinprotein interactions. These interactions are affected by binding of
small effector molecules (ATP, ADP, 2OG) 85, 86 and/or by posttranslational modification 78, 83, 87, although this is not a universally
conserved regulatory strategy.
As demonstrated by work in the laboratory of Alexander Ninfa using
the GlnB and GlnK proteins from E. coli, PII trimers can bind up to
three molecules of 2OG and ADP/ATP per trimer 85, 86. The binding of
ATP exhibits negative cooperativity and, upon ATP binding, up to
three molecules of 2OG are also able to bind. 2OG can only bind to
the PII protein trimer when ATP is present and, as ADP will compete
with ATP for the same binding site, higher ADP:ATP ratios will
displace 2OG, due to conformational changes in the proposed 2OG
binding site 88, 89. Recently a high resolution structure of Az. brasilense
GlnZ showed that 2OG is bound at the interface between the T-loops
of two adjacent PII protein subunits in the trimer. This binding site is
in close proximity to the ADP/ATP binding site, thus explaining the
effect of ADP:ATP ratios on 2OG binding 90. The characteristics of
ATP and ADP binding allow the PII trimer to act as an adenylate
energy charge sensor 85 and have actually been explored to create a
novel in vivo ADP:ATP ratio sensor 91. At the same time, 2OG can be
seen as a cellular carbon/nitrogen status indicator, with 2OG levels
usually inversely correlated with nitrogen availability 78, 92, 93. By the
binding of these different molecules PII proteins integrate diverse
metabolic signals and, by altering the three-dimensional structure of
20
the trimer and consequently the interactions with other proteins, they
transduce different signals.
PII proteins can also in many cases be post-translationally modified.
The most common form of modification in proteobacteria is the
uridylylation of a conserved tyrosine at position 51, as is the case for
E. coli GlnB 94, 95. Other types of modification, phosphorylation of a
conserved serine in cyanobacteria 96 and adenylylation in
actinobacteria (such as Corynebacterium glutamicum) 97, have also
been reported, usually as a reflection of nitrogen availability status 78,
87
. The additional complexity added with the possibility of one, two or
three modified subunits in the PII trimer leads to the (theoretical)
availability of some 56 different conformational and, consequently,
signalling states 98.
In R. rubrum three different PII paralogs have been described and
named GlnB, GlnJ and GlnK 99. The gene for the first paralog (glnB)
is co-transcribed with glnA, as is characteristic of GlnB-like PII
proteins. The transcription of the glnBglnA operon was shown to be
under the control of two different promoters, a nitrogen-responsive
σ54-dependent promoter in addition to the constitutive σ70 promoter
(see section 3.4.3. for more details). The final transcript is also
processed, yielding more glnA than glnB 100. glnJ and glnK are both
found in association and co-transcribed with amtB genes, amtB1 and
amtB2 respectively. Both GlnJ and GlnK belong to the GlnK-like
family. While the glnJamtB1 operon was shown to be regulated in
response to nitrogen availability, depending on the NtrBC system, the
glnKamtB2 operon does not respond to different nitrogen levels. The
R. rubrum glnK transcript is, in fact, the least abundant of all those
encoding PII proteins 101.
3.4.2. PII protein uridylylation
Modification of PII proteins adds an extra dimension of complexity
to regulation through protein-protein interactions. In proteobacteria
PII proteins are uridylylated by a bifunctional enzyme named
uridylyltransferase (UTase) / uridylyl-removing (UR) enzyme or
GlnD, encoded by glnD.
The uridylylation activity of R. rubrum UTase/UR requires the
binding of ATP and 2OG by the PII proteins, which will induce a
conformation favouring binding and uridylylation by UTase/UR.
Unlike the case for the E. coli enzyme, high glutamine concentrations
21
do not inhibit PII uridylylation. The deuridylylation reaction, on the
other hand, is highly stimulated by glutamine binding to UTase/UR,
with ATP and 2OG concentrations having little or no effect on activity
(Figure 4). The presence of equal amounts of unmodified PII protein
will inhibit the deuridylylation of modified PII in vitro 102. It was also
recently shown that varying ADP:ATP ratios can influence the
uridylylation activity, with higher ratios translating into less GlnJ
uridylylation 103.
Figure 4 – Regulation of R. rubrum PII uridylylation status by UTase/UR
3.4.3. Genetic regulation of nitrogen fixation
Nitrogen fixation is a demanding process for the cell. As nitrogenase
has a low turnover number, diazotrophic organisms produce large
amounts of this enzyme (as much as 20% of the cellular protein
content in some cases) in order to survive using dinitrogen as a
nitrogen source. In addition, owing to the oxygen-sensitivity of the
metal clusters, nitrogenase must be protected so as to avoid exposure
and inactivation by oxygen. These responses include growth
restriction to intracellular anaerobic conditions, metabolic and/or
physiological adaptations and also strict genetic regulation in response
to oxygen 54, 104.
Genetic control of nitrogen fixation in response to nitrogen
availability is a common trait in diazotrophs, with NifA as the
transcription factor responsible for regulation of nif gene expression in
Proteobacteria 54. The N-terminal of NifA proteins harbours a GAF
domain, which has been shown to be of great importance to signalling,
in some cases being able to bind small molecules 105. In A. vinelandii,
2OG binding by NifA modulates the activity of a second domain, the
AAA+ domain, in response to interaction with another regulatory
22
protein, NifL 106. However, this has not been demonstrated for other
nitrogen fixing bacteria, such as K. pneumoniae or Az. brasilense 54.
Even though strategies may differ, the outcome is always to regulate
expression and/or activation of NifA. In many organisms, such as K.
pneumoniae, NifA expression is regulated by the activation of the
two-component sensor-transducer NtrB/C system.
Under nitrogen limitation, the histidine kinase activity of NtrB
catalyzes the phosphorylation of NtrC and thus activation of
transcription of the nifLA genes. Expression and activation of NifA
induces transcription of other nitrogen fixation (nif) genes. In nitrogen
rich conditions, the phosphatase activity of NtrB catalyzes the
dephosphorylation of NtrC, inactivating nif gene transcription. The
activity of NtrB is in turn regulated by interaction with GlnB 107.
In R. rubrum strains expressing the unmodifiable variant Y51FGlnB, i.e. with a substitution at the conserved uridylylation site,
activation of nif gene expression is severely impaired and nitrogenase
activity is therefore greatly reduced. A strain in which the glnB gene is
deleted shows no activity and only very minor expression of
nitrogenase 108. The presence of GlnB and its uridylylation state are,
thus, important for nitrogenase expression in R. rubrum. However,
unlike the cases previously mentioned, the NtrB/C system is not
required for NifA expression, with R. rubrum ntrBC deletion strains
having 80% of the nitrogenase activity of the wild type cells 109. The
activity of NifA is directly regulated by interaction with GlnB and it is
the uridylylated form of the PII protein that will activate NifA and,
consequently, nif gene transcription 110. Therefore, the uridylylation
status of GlnB and the activity of UTase/UR are crucial for
nitrogenase expression.
In some organisms, such as Rb. capsulatus and Rb. sphaeroides,
there is a higher order of regulation, through the RegB/RegA twocomponent system. This system provides an additional layer of
complexity by responding to changes in the redox and energy status of
the cells. In Rb. capsulatus RegB/RegA have been implied in the
regulation of such diverse processes as photosynthesis, the transition
from aerobic to anaerobic respiration, carbon fixation and nitrogen
fixation 111. Also, in Rb. sphaeroides, mutations in the regB gene not
only block transcription of the cbb regulon (containing the genes for
RuBisCO and other enzymes of the Calvin-Benson-Bassham [CBB]
cycle for carbon fixation) but also of the nif operon. Furthermore,
strains carrying a non-functional cbbM gene (the gene for RuBisCO)
are able to derepress nitrogenase synthesis, even in the presence of
23
what would usually be inhibitory ammonium concentrations 112. The
same effect has also been observed in an R. rubrum RuBisCOdeficient strain, which hints at the existence of a global regulator
similar to RegB/RegA 112. However, no homologues for this particular
system have so far been found in R. rubrum thus still leaving this
question unanswered.
3.4.4. Metabolic regulation of nitrogen fixation
In addition to the control of nitrogenase expression, some organisms
also employ post-translational modification of dinitrogenase reductase
in order to control nitrogenase activity in response to extracellular
stimuli. Already in their first studies on nitrogen fixation in R. rubrum
Kamen and Gest had noticed that addition of ammonium would
temporarily inhibit hydrogen evolution in nitrogen fixing cultures 113.
Later studies showed that the period of inhibition has a direct
correlation with the amount of ammonium added 114. R. rubrum
nitrogenase can also be inactivated in response to the addition of
glutamine 115 as well as in response to light withdrawal 116 and, as in
the case of ammonium, this inactivation is reversible and due to posttranslational modification 115, 116, an effect known as nitrogenase
switch-off. Through the work of Neilson and Nordlund it was also
found that post-translational regulation of nitrogenase was lost upon
cell breakage as none of the conditions inducing switch-off in vivo had
the same effect on extracts 115.
The nature of the modifying group and the location of the
modification has been the subject of study of several groups 117-119. In
1985 the modification was shown to be an ADP-ribosylation in an
exposed and conserved arginine residue of NifH (Arg101), within an
equally conserved site in all NifH homologues 119. The modification
hinders the formation of the Fe protein – MoFe protein active
nitrogenase complex 120, 121, as it is located in the area that participates
in the docking to the MoFe protein 13 thus inhibiting electron transfer
and consequently nitrogen reduction. Modified Fe protein does not
compete with the unmodified form for the MoFe protein, when both
are present in nitrogenase activity reaction mixtures 120. Only one of
the two NifH subunits in the dinitrogenase reductase complex is
modified, giving modified Fe protein a characteristic double band
pattern in SDS-PAGE gels, a tool that allows researchers to easily
follow NifH modification in vivo as well as in vitro 116. The double
24
band pattern of post-translationally modified Fe protein has been used
extensively as a marker for identification of other diazotrophic
bacteria having a similar regulatory system.
The activating enzyme – DRAG
By the time the nature of the modifying group was discovered it had
already been known for some time that a protein component was
needed for reactivation of inactive nitrogenase. This “activating
factor” was specific for modified NifH, required MgATP and Mn2+ or
Fe2+ for activity and could be released from R. rubrum chromatophore
membranes using 0.5 M NaCl 122, 123. It was later purified and
characterized and shown to be a 32 kDa monomeric protein, which
was then named Dinitrogenase Reductase Activating Glycohydrolase
(or DRAG) 124.
DRAG is constitutively expressed 125 from the draTGB operon, an
operon containing also the gene for the inactivating enzyme (DRAT)
as well as a protein of so far unknown function (encoded by draB) 126.
The constitutive expression of both DRAG and DRAT is slightly
surprising as under nitrogen rich conditions the substrate for both of
these enzymes (Fe protein) will not be expressed. So far, no other
activities are known for DRAG or DRAT in addition to the regulation
of nitrogenase activity.
DRAG was purified and characterised and, despite initial reports that
claimed DRAG to be an oxygen-sensitive enzyme, this was later
shown to be merely an artefact of the preparation 127. As the name
indicates, DRAG catalyzes the removal of the ADP-ribose group from
dinitrogenase reductase, with a Km of 74 µM 124. Recently, a highresolution structure of R. rubrum DRAG was obtained. A reaction
intermediate using ADP-ribose could also be resolved to high
resolution and provided excellent insights into the reaction mechanism
of this enzyme 128.
The inactivating enzyme – DRAT
Soon after the identification of the modifying group Paul Ludden and
co-workers purified the enzyme responsible for the inactivation
activity to near homogeneity from R. rubrum cells and proposed the
name Dinitrogenase Reductase ADP-ribosylTransferase (DRAT) 129.
The enzyme was shown to be a monomeric protein of about 30 kDa
and, like DRAG, it is present in very scarce amounts in the cell 129.
25
DRAT was shown to be highly specific for the Fe protein 129 and to
prefer the oxidised form. However, and unlike DRAG, air-oxidized Fe
protein is not a substrate for DRAT and all activity assays must,
therefore, be performed under anoxic atmosphere 130.
DRAT modification of R. rubrum Fe protein requires an ADP-ribose
donor, with (β-)NAD+ being the most physiological supplier, with a
Km of 2 mM 129, even though other molecules, such as nicotinamide
hypoxanthine dinucleotide or etheno-NAD can substitute for NAD+ in
vitro 130. NAD+ is required for DRAT-Fe protein interaction 131, as
well as ADP and it has been speculated that ADP binding to Fe
protein regulates modification by DRAT 131, although, as we shall see
later on, this may not be the case.
Regulation of the switch-off effect
As DRAT activity requires NAD+ and DRAG activity will cleave the
ADP-ribose group from the Fe protein, the two enzymes should not be
active at the same time, in order to avoid depleting the cellular NAD
pool. Indeed, both DRAG and DRAT are subject to post-translational
control. However, the nature of the signals regulating their respective
activities is still, even after more than 30 years, the subject of ongoing
research. The signal transduction pathway seems to be kept
throughout a variety of organisms as was shown by switching the
draTGB operon between R. rubrum and Az. brasilense 132. In fact,
even organisms that do not possess such a post-translational regulation
of nitrogenase were able to modify the Fe protein in response to
switch-off stimuli once the dra operon was transferred to them 133, 134,
thus demonstrating the seemingly widespread nature of the signal
transduction pathway.
In R. rubrum the PII proteins GlnB and GlnJ were shown to play a
vital role in the regulation of this activity. Either one of the two
proteins can substitute for the other in the regulation of posttranslational modification of nitrogenase but none can be substituted
by GlnK in that role 99. The relevance of PII proteins for posttranslational regulation of nitrogenase has also been demonstrated in
other organisms, such as Rb. capsulatus 135 and Az. brasilense 136, 137.
DRAG regulation by membrane sequestration
As previously mentioned DRAG is found in association with the
membrane in both ammonium and in darkness switched-off R. rubrum
26
cells. Membrane sequestration is thought to be the mechanism by
which DRAG is inactivated during switch-off, as it will not allow
interaction with its subtrate (ADP-ribosylated-Fe protein). Recently
several advances have furthered our understanding of this mechanism
and shown that the ammonium/methylammonium transporter AmtB,
in complex with GlnK or GlnK-like proteins, is crucial for correct
regulation in ammonium switch-off.
The widespread genetic linkage observed for the amtB and glnK
genes 82 suggested the existence of a physiological link between the
two proteins and, as observed in E. coli and A. vinelandii, GlnK is
localized to the inner membrane in response to a variation in the
nitrogen status in an AmtB-dependent manner. In nitrogen-limited
cells GlnK is uridylylated and, even though both AmtB and GlnK
expression is highly induced, the two proteins do not interact until an
influx of nitrogen brings about the deuridylylation of the PII
protein.138. Thus, the UTase/UR control of the PII uridylylation status
allows efficient signalling of the nitrogen status, transmitted through a
differential localization of GlnK. Structures of this AmtB-GlnK
complex from E. coli are already available and have provided valuable
information on how GlnK regulates transport through AmtB 139, 140.
An interesting detail of these structures is that ADP was found in all
nucleotide-binding sites of GlnK. It was shown that AmtB-GlnK
complex formation in E. coli requires the presence of ADP and that
GlnK can be specifically released from AmtB-containing membranes
by washing with ATP and 2OG 141.
In R. rubrum GlnJ was also found to require AmtB1 for membrane
association 142 and, as in the case of E. coli, the complex can be
dissociated in the presence of Mn2+, 2OG and low ADP:ATP ratios 103,
143
. AmtB2, the other AmtB homologue, has no influence on either
GlnJ association to the membrane or nitrogenase switch-off 144. It
should at this point be noted that it is the unuridylylated form of GlnJ
that binds to AmtB1 and that the conditions required for complex
dissociation (MnATP and 2OG) are also the conditions stimulating
uridylylation of PII proteins, as previously discussed.
Upon ammonium switch-off in R. rubrum, DRAG is localized to the
chromatophore membrane and this association was shown to be
dependent on the presence of AmtB and GlnJ. As a consequence,
AmtB1 mutants have impaired ammonium switch-off 142. Membrane
sequestration effectively separates DRAG from ADP-ribosylated-Fe
protein, allowing nitrogenase switch-off. AmtB-dependent membrane
27
sequestration of DRAG was also seen in Az. brasilense 145. However,
the specific membrane partner for DRAG association is still not yet
clearly identified. DRAG could bind “on the side” of the AmtB1-GlnJ
complex, interacting with the interface between the two proteins or
only with AmtB1. A third possibility is that there is a still unknown
membrane partner in the vicinity of the AmtB1 protein with which
DRAG could also interact.
Nitrogenase is also modified in response to energy signals, namely
darkness in R. rubrum and Rb. capsulatus 146 and anaerobiosis in Az.
brasilense 147. However AmtB1-deficient R. rubrum strains respond in
a different manner to darkness, depending on the nitrogen source used.
Indeed, when using glutamate-grown nif-derepressed R. rubrum
strains lacking AmtB1, the darkness switch-off response is perturbed,
with nitrogenase activity being reduced to ~50% 144. However, when
grown diazotrophically, darkness switch-off is only marginally
affected in R. rubrum amtB1 mutant strains 101. As recently described,
PII proteins are differentially uridylylated upon darkness-induced
switch-off, depending on which nitrogen source is used. In glutamategrown cultures PII proteins will be deuridylylated in response to
darkness whereas diazotrophically-grown cultures will maintain PII
proteins uridylylated 148. This difference could eventually play a
significant role in the control of nitrogenase darkness switch-off in
these two conditions as an uridylylated GlnJ will not associate to
AmtB1 and, thus, DRAG binding to the membrane could be partially
hindered. However, as DRAG can still be found to a large extent by
the membrane upon darkness switch-off in diazotrophic conditions,
even in a strain lacking AmtB1 – in which (uridylylated) GlnJ is only
found in the cytosolic fraction 101 – the darkness switch-off response
in R. rubrum under these conditions may effectively include an as yet
unknown membrane partner.
A similar effect is observed in AmtB-deficient Rb. capsulatus
strains, with switch-off being affected only in response to ammonium
but not to darkness 146. It could be speculated that another (presumably
integral or membrane-associated) protein is involved in DRAG
membrane sequestration. This putative protein could be involved in
sensing energy changes in the cell and, by interaction with DRAG
and/or AmtB, regulate the darkness switch-off response.
28
DRAT regulation by protein-protein interaction
Whereas some of the details regarding DRAG regulation in
nitrogenase switch-off are already known, the mechanisms controlling
DRAT are much less understood. DRAT fractions obtained from R.
rubrum cultures were shown to be able to modify Fe protein in vitro
129
. However, this “purified fraction”, as well as those used in later
studies based on the same purification procedure, contains a low-mass
contaminant, which was at the time considered unimportant for the
regulation of the ADP-ribosylation system. On the other hand,
attempts to remove this contaminant fraction lead to “unacceptable
losses” of DRAT activity 129.
It was demonstrated by yeast-two hybrid studies that DRAT and
GlnB from both R. rubrum and Rb. capsulatus can form a complex 149,
150
. This putative complex was further studied using purified GlnB and
DRAT proteins from Az. brasilense. DRAT is a notoriously unstable
protein and Az. brasilense DRAT over-expression in E. coli usually
results in the formation of inclusion bodies. However, co-expression
of GlnB with DRAT was found to decrease the formation of these
inclusion bodies and stabilize DRAT in solution 151. In vitro studies
using the purified proteins from Az. brasilense showed that DRAT
interaction with GlnB is stabilized by the presence of ADP and,
conversely, destabilized by addition of ATP and 2OG 151. It should be
noted once more that the same conditions that result in Az. brasilense
DRAT-GlnB complex dissociation in vitro allow release of GlnJ from
R. rubrum chromatophore membranes and uridylylation of PII
proteins. Modulation of the interaction is most probably due to
binding of the small effector molecules by GlnB with the consequent
changes in three-dimensional structure of the PII trimer regulating its
interaction with DRAT. Huergo and co-workers have speculated that
this interaction with GlnB could regulate DRAT activity in vivo in
response to fluctuations in the ADP:ATP ratios and 2OG
concentration, which would also explain the afore-mentioned need for
ADP in the DRAT activity assay 129. We shall discuss the influence of
GlnB on DRAT activity in a later section.
The redox state of the pyridine nucleotide pool was also previously
implicated in the regulation of the switch-off effect, with NAD+ being
able to induce Fe protein ADP-ribosylation and consequent switch-off
152, 153
. As NAD+ is the preferred substrate for DRAT it could be that
induction of switch-off is a reflex of the higher abundance of one of
the substrates (NAD+). Also, a change in the NAD+:NADH ratio (and,
29
thus, the cellular redox state) could be sensed by other protein(s)
influencing DRAT or DRAG activity. Indeed, a decrease in the
intracellular NAD(P)H concentration was observed when R. rubrum
cells were subjected to different switch-off effectors 152.
Other strategies for nitrogenase switch-off
So far we have only discussed post-translational regulation of
nitrogenase by ADP-ribosylation. However, nitrogenase has also been
shown to be regulated by direct interaction with proteins of the PII
family in Methanococcus maripaludis. In this methanogenic archaea
the PII protein homologues NifI1 and NifI2 form a complex that
directly interacts with the NifDK nitrogenase subunits and effectively
interrupts electron flow in response to ammonium, causing switch-off
84, 154, 155
. As these PII proteins are highly spread among archaea and
anaerobic bacteria it has been suggested that this may be a more
common regulatory strategy than initially though 84.
Some studies in cyanobacteria have also hinted at the possibility that
at least in some cyanobacteria nitrogenase may be regulated be posttranslational modification 156, 157. However, further studies are
necessary as substantial data is still missing to validate those claims.
3.4.4. Genetic and metabolic regulation of nitrogen assimilation
Nitrogen assimilation, just as nitrogen fixation, is the subject of tight
regulation, both at the transcriptional and post-translational level.
Unlike nitrogen fixation, the assimilation process must be present at
all times, to ensure the cells are supplied with the preferred
biologically usable forms of nitrogen – amino acids, usually glutamate
and glutamine. The process of nitrogen assimilation through the
GS/GOGAT pathway is so fundamental that E. coli cells devote a
large percentage (ca. 15%) of their overall ATP supply to this process
alone 69.
Genetic control of glutamine synthetase
Under nitrogen rich conditions, the gene for GS, glnA, is transcribed
from σ70-dependent promoters, the “house-keeping” RNA polymerase
form. On the other hand GS expression is induced in nitrogen
30
deficiency 78. The Ntr system has been implicated in the regulation of
expression of the glnA gene in several organisms, including E. coli 69,
R. rubrum 100 and K. pneumoniae 158.
In R. rubrum, unlike in enteric bacteria 159, the glnB and glnA genes
are together in a functional glnBA operon, which was also shown to be
under the control of both σ70- and σ54-dependent promoters, thus
responding to nitrogen availability 100, 160.
Post-translational control of glutamine synthetase
Glutamine synthetase is a known target for feedback regulation by
nitrogen-containing molecules, such as alanine, serine, histidine,
tryptophan and others. In the case of the E. coli the different inhibitors
can only induce partial reduction of activity, in a process known as
cumulative inhibition 161. The R. rubrum enzyme is also sensitive,
albeit with some differences in the extent of inhibition, to some of the
same feedback inhibitors as its E. coli counterpart 162.
GS can also be regulated by post-translational modification.
Adenylylation of a conserved tyrosine residue (Tyr397 in the E. coli
enzyme) in each of its twelve subunits decreases the activity of GS
sequentially, with increasing levels of modification decreasing GS
activity 163. In fact, GS adenylylation in E. coli was the first described
example of the use of nucleotydylation as a means to control enzyme
activity 164 and was since then found to occur in a variety of
organisms, including R. rubrum 162, 165-168. Both the addition and
removal of the AMP group are catalysed by the same bifunctional
enzyme, adenylyltransferase (ATase). This enzyme is regulated by
interaction with the PII proteins and, as the uridylylation state of the
PII proteins is also controlled by the concentrations of 2OG and
glutamine, one can easily correlate GS activity with that of UTase/UR.
In R. rubrum any of the three (non-uridylylated) PII proteins can
stimulate the adenylylation activity 99, 108 but this reaction, unlike the
case for the E. coli enzyme, does not require the presence of glutamine
93
. PII-UMP and 2OG inhibit the adenylylation reaction although 2OG
inhibition can be relieved by high ADP:ATP ratios in vitro 103.
Regulation of the deadenylylation reaction, however, does not require
neither PII-UMP nor 2OG, with the only requirement being Pi (Figure
5). This makes deadenylylation the standard activity for R. rubrum
ATase, with 2OG concentration and ADP:ATP ratios controlling the
31
shift between both activities, through binding to the PII proteins (and,
indirectly, by changing their uridylylation status) 93, 103.
Figure 5 – Regulation of R. rubrum GS post-translational modification by ATase
GS activity is usually measured by the colorimetric γglutamyltransferase assay 169. This assay can be used to estimate the
number of modified E. coli GS subunits by using either Mn2+ (in
which case both modified and unmodified forms are active) or Mg2+
(when only unmodified GS will be active) in the reaction and
determining the relation between the two 170. In the case of the R.
rubrum enzyme such estimation is not possible as the behaviour of the
modified and unmodified forms of GS in respect to Mn2+ or Mg2+
differs from that of E. coli 168, 171. However, a decrease in GS activity
using this assay can nevertheless be correlated with adenylylation and
vice-versa 162.
Why is the post-translational control of GS activity so important that
it would require such an elaborate control system? One could argue
that regulation of GS expression should suffice to adapt to different
nitrogen supplies. However, control of expression provides a very
crude means for regulation and does not allow fine-tuning of
enzymatic activities in response to rapid changes in the environment.
In E. coli a sudden influx of ammonium causes rapid consumption of
90% of the ATP pool and a 20-fold increase in glutamine
concentration before GS activity is regulated 172 and this would be an
unsustainable condition for the cells if it was maintained for very long.
Likewise, it was recently shown that deleting the gene for ATase
(glnE) results in severe growth defects in R. rubrum and accumulation
of secondary mutations that ultimately lead to low GS activity 173. GS
adenylylation could then have evolved in organisms in which GS has
32
a high specific activity as a means to protect the intracellular pools of
critical metabolites.
3.5. 2-oxoglutarate, a key metabolite
Whether discussing nitrogenase expression or regulation, PII protein
modification or GS adenylylation in R. rubrum, 2OG is a ubiquitous
metabolite, directly or indirectly related to all of these processes. Its
concentration is usually inversely proportional to the nitrogen status of
the cell and usually this is perceived through the action of the PII
proteins. 2OG is an intermediate of the Krebs cycle but, due to its role
in the process of nitrogen assimilation and, in some cases, carbon
fixation 174, it occupies a very central role in the cellular metabolic
routes. In vitro, high 2OG concentrations stimulate UTase/UR to
uridylylate PII proteins and this not only activates NifA and nif gene
expression but it also stimulates DRAG release from the
chromatophore membranes, at the same time that GS adenylylation by
ATase is inhibited. The concentration of 2OG was shown to be higher
in nitrogen fixing R. rubrum cells than in cultures grown in nitrogen
rich conditions 93, correlating the in vitro data with physiological
effects in vivo. Addition of ammonium to R. rubrum nitrogen fixing
cultures was shown to cause a temporary increase in the intracellular
concentration of glutamine 116, due to the increased flux through the
GS/GOGAT pathway, at the expense of 2OG. The combined effect of
the “glutamine surge” and 2OG pool depletion will activate
deuridylylation of PII proteins, resulting in inhibition of nif gene
expression, GS adenylylation and GlnJ (as well as DRAG) association
to the membrane. Even though this is an over-simplified view of the
regulatory schemes controlling nitrogen fixation and assimilation in R.
rubrum, as other factors such as the redox balance and ADP:ATP
ratios must also be taken into account, it allows us to have a global
view of the interplay between the different processes in the cell.
33
4. An overview of the field of proteomics
After the explosion of data deriving from the many gene and genome
sequencing projects in the late 1990’s, the term “proteomics”, a
contraction of “protein” and “genomics”, arose as an analogy to the
study of the genome 175. However, the proteome is a much more fluid
concept, as it refers to the entire set of proteins present in an organism
or specific part of an organism, at a given time, under a certain set of
conditions. As it can be easily understood, unlike the genome, which
is usually a more static entity, the proteome will vary according to the
environmental and intracellular conditions and, in multicellular
organisms, different cells will have different proteomes, even though
their genetic material should be the same. Also, if one adds the extra
dimensions of post-translational modification and protein-protein
interactions, the study of the proteome assumes an even more
considerable breadth 176.
In practical terms, proteomic analysis is usually synonymous with
techniques designed to facilitate the identification of proteins in a
given sample and quantitate differences in their relative abundance. It
should however be noted that this is a rather reductive view of the
field of proteomics as, for example, studies regarding in vitro
characterization of the interaction between different proteins in a
complex can also be encompassed by the definition of “proteomics”.
Nevertheless, and taking the first description of practical proteomics,
two major branches are today distinguished, based on the platform
used to perform the study – namely, gel-based and gel-free
proteomics.
4.1. Gel-based proteomics
One of the most widely spread techniques in any life sciences
laboratory today is that of the polyacrylamide gel electrophoresis, with
its many variants. The most common technique is that employing an
anionic denaturing detergent, sodium dodecylsulphate or SDS, which
allows migration of the target proteins through the polyacrylamide gel
matrix upon application of an electric field and their separation solely
based on the relative mass of each protein – a technique known as
(one-dimensional) SDS-PAGE. In this way, complex mixtures of
proteins can be easily separated, allowing the relative quantification of
each band, usually by staining the protein bands with a dye 177. This is
34
a relatively fast and inexpensive technique but it also has its
drawbacks. Proteins of similar mass or the same protein with different
post-translational modifications, for example, will migrate closely
together, sometimes in the same band and, thus, this technique has a
relatively low separation capacity as well as low throughput.
In order to increase resolution for many polypeptides in the same
sample, several techniques emerged, making use of two different
intrinsic properties of proteins. In this way, proteins could be
separated according not only to their (unitary) mass but also with
respect to their isoelectric point (pI) or the overall mass of the
complex in which they could be found, for instance.
Two-dimensional isoelectric focusing (IEF) / SDS-PAGE (2D-PAGE)
The first attempts at establishing a two-dimensional acrylamide gel
electrophoresis separation based on the proteins’ pI and mass were
performed using acrylamide gels cast inside thin cylindrical tubes,
containing zwitterionic molecules, termed ampholites, used to
generate a pH gradient throughout the gel, upon application of an
electric field. Protein samples loaded onto the gel will migrate to the
zone where they have no net charge, where they will have reached
their pI. The first dimension gel was then removed from the tube and
loaded on a regular SDS-PAGE gel for the second dimension
separation 178-180. However, this first technique had some
reproducibility problems related to the pH gradient in the first
dimension 181. This posed a major hurdle until the development of the
immobilized pH gradient (IPG) technique. Instead of “free”
ampholytes, the molecules used to create the IPG – later termed
“Immobilines” – have acrylamido groups that, upon IEF gel casting,
are covalently cross-linked to the gel matrix, rendering the pH
gradient stable and reproducible 182. The mass production of these
first-dimension gels reduced variability even further and allowed
increased output on the technique, even though it is still rather labourintensive.
Already in the first attempts, 2D-PAGE was shown to be a powerful
technique, allowing the separation of hundreds or even, in some cases,
a few thousands of proteins from a complex sample into independent,
discrete spots in a single gel 183. With the development of gel image
analysis algorithms, it was now possible to instantly quantify changes
in sample sets containing several hundreds of proteins. As posttranslational modifications induce a shift in the pI, this technique can
also monitor modifications as well as discriminate between different
35
protein isoforms. Nevertheless, 2D-PAGE also has its disadvantages
and flaws, the most notorious being the under-representation of
membrane proteins, very high and very low mass proteins or those
with alkaline pI as well as the “shadowing” of low-abundance proteins
by other more abundant proteins in the sample 184.
Despite considerable advance in this area, many membrane proteins
are insoluble in the detergent/chaotrope mixture usually used to
prepare protein samples for the first dimension of 2D-PAGE and, of
those that can be solubilised, many precipitate at or near their pI
during the IEF run 185. Other specialized gel systems have been
devised to address this problem, using different combinations of
denaturing detergents 186 or, in order to analyse the formation of
protein complexes in the membrane, a combination of gentle
detergents, extracting proteins and complexes in near-native
conditions, followed by a denaturing SDS-PAGE second dimension, a
technique that will be described in more detail below.
Despite its shortcomings, 2D-PAGE is still one of the most widely
used quantitative proteomic techniques in laboratories worldwide and,
in combination with mass spectrometry, is still an efficient, reliable
and relatively economical tool.
Two-dimensional Blue Native (BN) / SDS-PAGE (2D BN/SDS-PAGE)
One of the most common systems used for analysis of membrane
protein complexes is the one developed by Schägger and von Jagow in
1991, termed Blue Native (BN) electrophoresis. This system allows
the separation of membrane proteins in (near-)native complexes,
extracted from the membrane using mild, non-ionic detergents, such
as dodecylmaltoside (DDM), Triton X-100 or digitonin 187. The name
of the technique comes from the usage of the Coomassie Brilliant
Blue (CBB) G-250 dye in the sample and cathode buffers. The
binding of the dye to the protein complexes results in an overall
negative surface charge, allowing them to migrate in the gel according
to mass only.
The individual components of each complex can be analysed using a
second dimension denaturing gel run, as for the 2D-PAGE system.
The overall result is a two-dimensional map that not only allows
quantification of changes in the membrane and membrane-associated
proteome but also allows the study of protein-protein interactions – all
in the same system.
As can be perceived, the system depends on the efficiency of
extraction of the membrane protein complexes by the chosen
36
detergent. Even if a mild detergent is used, the addition of the CBB G250 dye, though vital for the technique, may also cause disaggregation
of some more loosely bound protein subunits 188.
Despite its very informative characteristics 2D BN/SDS-PAGE is a
time-consuming, labour-intense and relatively low-throughput system,
just as other gel-based proteomics techniques.
4.2. Gel-free proteomics
The fastest-growing trend in proteomic studies is the use of gel-free
systems, based on on- or off-line coupling of liquid chromatography
and mass spectrometer systems. These systems offer several
advantages when compared with the “traditional” gel-based methods:
identification and relative quantification of (mostly) all the proteins
present in very complex samples, higher sensitivity, reproducibility
and, due to automation, higher throughput 189. These techniques
usually involve fractionation using different chromatographic
platforms of the peptide mixture resulting from the digestion of all the
proteins in the sample and subsequent identification (and, frequently,
relative quantification) of the proteins present in the sample using
mass spectrometry. By analogy to the technique used to sequence
large DNA sequences, this kind of approach is usually called
“shotgun” proteomics 190. Frequently, in order to allow relative
quantification of the samples, the peptides are tagged using, for
example, different radioactive isotope-containing molecules. Even
though these techniques present several improvements in the limit of
detection and analysis throughput, the implementation cost is usually
much higher than that of the gel-based techniques, as they require a
much more sophisticated setup, both at the hardware as well as at the
software level 189. As such, they are still too costly to be as widespread
as the more common gel-based techniques.
4.3. Mass spectrometry
If it is true that the improvements in separation techniques allowed
proteomics to grow in “size”, i.e., in resolution and in number of
proteins individually separated in a sample, it was only with the
implementation of mass spectrometry, in its different varieties, that
quantitative proteomic studies could finally become a robust tool for
research. Prior to the advent of protein mass spectrometry, protein
identification was achieved by the use of either specific antibodies or
37
Edman degradation. However, even though it is a highly reliable
method, Edman degradation has some severe limitations – it is very
time-consuming and expensive and N-terminal modifications will
limit its applicability 191.
Mass spectrometers usually have three main components: the ion
source (with ion accelerators and optics), the mass analyser modules
and the integrated data-processing software. The adaptation of mass
spectrometry ionization techniques to the ionization of peptides and
proteins resulted in the introduction of “softer” ionization methods
such as “matrix-assisted laser desorption/ionization” (MALDI) or
“electrospray ionization” (ESI). These can ionize peptides and even
whole proteins without extensive degradation. MALDI uses a laser to
ionize and desorb dried samples mixed with a matrix, creating a cloud
of usually singly charged ions. ESI, and its successor, nanospray
ionisation, ionize peptides and proteins directly from solution, using a
thin capillary to which high voltage is applied, resulting in the
formation of a conic cloud of vaporised peptide ions. ESI and
nanospray allow analysis of multiply charged ions and, coupled to low
mass limit spectrometers, facilitate detection of high mass molecules
191
.
The mass analyser is the “core” of the mass spectrometer and many
different types exist. One of the most common and widely spread is
the “time-of-flight” (ToF), based on the time required for the different
peptides to “fly” from one end to the other of the so-called “flight
tube”. Other mass analysers detect mass:charge ratio (m/z) of the
peptides, either by the usage of the intrinsic resonance frequency (as
ion traps, Orbitraps and ion cyclotron resonance analysers) or
detection of m/z stability, as in the case of ion quadrupoles (Q). Many
mass spectrometers have hybrids of two or more of these analysers –
an example is the ESI-Q-ToF mass spectrometer 192.
MALDI-ToF
One of the most basic and widely used setups is that where a MALDI
ionization source is coupled to a ToF mass analyser – the MALDIToF mass spectrometer. Although it can also be coupled to a nanoliquid chromatography (LC) system to enhance sensitivity and
separation of different peptides in a sample, this kind of spectrometer
is commonly used off-line, in combination with gel-based proteomics.
Gel spots containing the protein of interest are digested using a
specific protease (most commonly trypsin) and the peptide digest is
38
analysed off-line in the MALDI-ToF. The resulting spectrum contains
a combination of the different monoisotopic peptide masses resulting
from the protein digestion and is compared with a database containing
all the theoretical mass lists for all the proteins in a specific set,
derived from genome sequences. This identification process, as it
generates a characteristic mass list for each protein, is termed “protein
mass fingerprinting” 192. Even though it is one of the most widely
spread and used mass spectrometers around the world, the MALDIToF has many limitations. Its relatively low sensitivity and resolution,
requiring the collection of a large number of shots for a good signalto-noise ratio, hinder identification of low-abundance peptides and, as
it uses laser pulses to ionize peptides, there is a high degree of
variability between each shot.
Orbitrap
Unlike the “pulsed analysis” of the MALDI-ToF, other mass
spectrometers physically “trap” the peptides in the sample to analyse,
allowing a faster workflow, higher throughput, versatility and
sensitivity. One of the most recent developments in this field is the
Orbitrap, an ion trap whose main feature is a spindle-shaped central
electrode. Commonly, a linear ion quadrupole trap is placed in front of
the Orbitrap (LTQ-Orbitrap) and samples, injected from on-line LC,
are ionised using ESI or nanospray. The inclusion of collision cells
allows peptide sequencing, resulting in an instrument with very high
sensitivity and almost unrivalled accuracy (0.2 ppm at signal-to-noise
ratio of >10.000) 193. The Orbitrap is a very powerful new tool in the
field of proteomics albeit still at a relatively high cost, which hinders
its more widespread usage.
In the first part of this thesis we have approached some of the basic
concepts regarding nitrogen metabolism in diazotrophic bacteria, as
well as some of the technical aspects regarding proteomic studies. We
shall now discuss some of the most recent findings regarding the
adaptation of R. rubrum to different nitrogen availability conditions as
well as some more detailed aspects of nitrogen metabolism regulation.
39
II. Adaptation to different nitrogen
conditions
1. The interconnection between nitrogen fixation and
carbon fixation pathways
How does R. rubrum respond to a shift in nitrogen availability –
from nitrogen rich to nitrogen fixing conditions? We have already
discussed how nitrogenase is usually only expressed when nitrogen is
limiting and this is, naturally, the hallmark of nitrogen fixation. The
GS/GOGAT cycle enzymes as well as GDH are also differentially
regulated. The expression of these enzymes is known to respond to the
cellular nitrogen status, as previously discussed, and the effects seen
on R. rubrum in nitrogen fixing conditions (I) correlate well with
these previous observations. However, there are other effects that
occur upon a change in the nitrogen status in the cell. One of the most
striking is the down regulation of several enzymes involved in
different carbon fixation and utilization pathways in nitrogen fixing
conditions. These include ribulose–1,5–bisphosphate carboxylase /
oxigenase (RuBisCO), some enzymes of the reductive tricarboxylic
acid cycle and others proteins, involved in the synthesis of the storage
compound poly-hydroxybutyrate (PHB) (I).
RuBisCO
The regulation of the CBB cycle enzymes has previously been shown
to be linked to nitrogenase synthesis, with R. rubrum RuBisCO
mutants derepressing nitrogenase synthesis even in the presence of
ammonium 112. Conversely, it was also demonstrated that mutations in
the FixABCX pathway would induce up regulation of RuBisCO in
diazotrophically grown R. rubrum cultures 56. Thus, RuBisCO can be
seen not only as an enzyme catalysing carbon fixation but also as an
“electron sink”, diverting the excessive reducing power that can no
40
longer be used by nitrogenase under those conditions, in order to
maintain the redox balance in the cell.
Such a correlation between RuBisCO and nitrogenase has been
previously shown not only in R. rubrum but also in other purple nonsulphur nitrogen fixing bacteria, such as Rb. capsulatus, Rb.
sphaeroides and Rhodopseudomonas palustris 112, 194, 195. It has been
proposed that the RegA/RegB system could sense changes in the
cellular redox state and regulate the activity of CbbR and NifA
accordingly. The transmembrane region of RegB is thought to sense
redox changes, although the exact nature of the signal and its sensing
mechanism is still not understood 196. Even though there is no
homologous system in R. rubrum it is clear that a similar “redox
sensor/transducer” mechanism must be present, in order to regulate
not only the expression of the cbb and nif operons but also of other
processes, such as the PHB synthesis or regulation of the TCA cycle
enzymes. Interestingly, artificially lowering the intracellular 2OG pool
in nitrogen fixing conditions can also influence nitrogenase and
RuBisCO expression levels. As the PII modification status is
perturbed in these conditions, efficient NifA activation is also
hindered, resulting in lower levels of nitrogenase. It could be that the
lower levels of nitrogenase under such conditions lead to an imbalance
in the redox state and that this, in turn, induces the expression of
alternative electron sinks, e.g. RuBisCO (II).
The Reductive TCA cycle
Described for the first time in 1966 in Chlorobium thiosulfatophilum,
the reductive tricarboxylic acid (RTCA) cycle faced severe criticism
before it was finally accepted as an independent means to fix carbon
in anaerobic bacteria 174. This pathway uses specific enzymes, 2oxoglutarate synthase and pyruvate synthase, both requiring the
reducing power of reduced ferredoxins for catalysis, effectively
reverting the “common” TCA cycle. Coupled to this cycle, another
specific enzyme, ATP-dependent citrate lyase, couples CO2 fixation
by the RTCA to acetyl-CoA production 197. This system was also
found to exist in R. rubrum, with activity measurements
demonstrating the presence of both 2OG synthase, pyruvate synthase
and citrate lyase in photolithoautotrophically-grown R. rubrum
cultures. However, no citrate lyase activity could at the time be
demonstrated in photoheterotrophic conditions and it could be that the
RTCA cycle is incomplete under such conditions 198, perhaps being
present only as a means to regulate redox poise. In fact, 2OG synthase
is down regulated in nitrogen fixing conditions, mirroring the
41
behaviour of RuBisCO (I), and this could indicate that this cycle is
also subjected to redox regulation.
2. Keeping the (redox) balance
Differential regulation of both carbon and nitrogen fixation and
utilization pathways is necessary in order to maintain the carbon-tonitrogen balance in the cell but, moreover, it also allows control over
the cellular redox balance. Both nitrogen fixation and carbon fixation
require reducing equivalents and, therefore, it is important for the cell
to be able to regulate flow through each of these pathways, so as not
to generate an imbalance in the reductant pools. We have already seen
how different nitrogen availability levels affect the soluble proteome,
namely the level of expression of nitrogenase and RuBisCO (I and II).
However, such changes also influence the chromatophore membrane
components. FixC, for instance, was shown to associate to the
chromatophore membrane and the fixABCX operon is subjected to
regulation by nitrogen availability 56. Although the system developed
for membrane complex analysis did not allow the identification of any
membrane protein components associating to FixC, the higher level of
FixC in the chromatophore membrane samples of nitrogen fixing cells
(III) is in accordance with those previous studies.
The succinate dehydrogenase complex (Sdh) is up regulated under
nitrogen fixing conditions (III). It was previously shown that under
photoheterotrophic conditions the TCA cycle is essential for the
maintenance of normal levels of nitrogenase activity in R. rubrum,
perhaps generating the reducing equivalents needed for the nitrogen
reduction activity. Specific inhibition of the TCA cycle using
fluoroacetate results in decreased nitrogenase activity, an effect that
can be reverted by addition of NAD(P)H 199.
In E. coli the expression of Sdh and other TCA cycle enzymes is
regulated by the ArcA/B and FNR sensor/transducer systems 200, 201.
These systems respond to changes in carbon source and oxygen
availability and it was shown that the ArcA/B system can directly
sense variations in the quinone pool redox state 202, 203. Under reducing
conditions ArcB, an integral membrane protein sensor kinase, is
activated and phosphorylates ArcA, the cytosolic effector. This
enhances binding affinity of ArcA for the promoter regions of genes
encoding TCA cycle and respiratory chain components. Oxidising
conditions revert this phosphorelay system 204 and, as ArcA has an
inhibitory effect on the expression of the TCA cycle enzymes under
42
reducing conditions, the redox poise will in this way regulate their
expression level 205.
It was recently shown in R. rubrum that chromatophore membrane
biosynthesis was regulated by the redox state of the quinone pool.
Transfer to microaerophilic conditions and reduction of the quinone
pool induces chromatophore biogenesis and it was speculated that
sensing of the quinone pool redox state could be a more general signal
than previously thought 206. Searches on the R. rubrum genome for
homologues of either ArcA/B or FNR were unsuccessful. However, as
changes in the cellular redox state are able to influence the proteome
and, given that the quinone pool is able to reflect these fluctuations, it
is possible that a system with similar characteristics to ArcA/B, FNR
or RegA/RegB is also present in R. rubrum. As nitrogenase activity
will require a great part of the cellular reducing power, this could
induce an oxidation of the quinone pool and perhaps, through either
direct or indirect sensing of these changes, bring about the activation
of a signal relay system, ultimately regulating RuBisCO and TCA
cycle enzymes, among other targets (Figure 6). Such a system could
be considered a global regulator, regulating transcription/activation of
NifA, CbbR and other regulators. In this way changes in the nitrogen
availability would be reflected in the cellular redox balance and, to
maintain redox homeostasis, in both nitrogen and carbon fixation and
utilization pathways, by regulation of the proteome.
Figure 6 – Proposed mechanism for proteome regulation in different nitrogen
conditions, through sensing of changes in the redox state of the quinone pool.
43
III. Post-translational regulation of
nitrogenase activity – guarding the guards
1. Regulation of ammonium switch-off
Addition of ammonium to nitrogen fixing R. rubrum cultures
induces, as we have discussed, DRAG and GlnJ membrane
association in an AmtB1-dependent manner. In this way DRAG is
sequestered and, as it is physically separated from its substrate, it is no
longer able to remove the ADP-ribose moieties from the modified Fe
protein. But how is DRAT activity regulated upon ammonium switchoff?
It had previously been proposed, based on yeast two-hybrid and
mutagenesis studies, that R. rubrum DRAT and GlnB could form a
complex 149. Co-expression of N-terminally tagged R. rubrum DRAT
and GlnB in E. coli showed that the two proteins can, in fact, form a
stable complex in vivo. This complex, once isolated, is stabilized by
the presence of ADP and destabilized by low ADP:ATP ratios and the
presence of 2OG (IV). These results are in agreement with those
previously reported for the Az. brasilense DRAT-GlnB complex 151.
Intracellular 2OG concentrations are lower in nitrogen rich than in
nitrogen fixing cells 93 and, as such, the sensitivity of the DRAT-GlnB
complex to 2OG concentration could be reflected on the regulation of
DRAT activity. Indeed, the DRAT-GlnB complex was found to be the
physiologically active form, being able to modify R. rubrum Fe
protein in the presence of ADP and β-NAD+. Lower ADP:ATP ratios
and the presence of 2OG destabilize complex formation and,
consequently, inhibit DRAT activity (IV). Considering the difference
in 2OG concentrations in vivo between nitrogen fixing and nitrogen
rich conditions it could be extrapolated that addition of ammonium
will induce a higher flux through GS/GOGAT (and GDH), decreasing
the internal pool of 2OG. This elevated flux through GS could also
lead to a momentary increase in the ADP:ATP ratios, until GS is
modified. The lower concentration of 2OG and the possible increase
44
in ADP concentration, through binding to GlnB, will stabilize the
DRAT-GlnB complex and, thus, lead to Fe protein modification. It
should be kept in mind that these conditions also lead to PII protein
demodification and the association of GlnJ and DRAG to the
chromatophore membrane.
As the ammonium is consumed, the 2OG pool will once again
increase and destabilize the DRAT-GlnB complex, leading to DRAT
inactivation. At the same time, the elevated concentrations of 2OG
will also stimulate PII modification and both GlnJ and DRAG are
released from the membrane, reactivating modified Fe protein (Figure
7).
Figure 7 – Proposed mechanism for regulation of post-translational modification of
nitrogenase in R. rubrum in response to ammonium. E. coli AmtB1 and GlnK
atomic structures (PDBID: 2NUU) as well as R. rubrum DRAG (PDBID: 2WOE)
were generated using PyMol 1.0 and the respective PDB coordinates.
In this way, intracellular 2OG concentrations will have a big impact
on the regulation during ammonium switch-off, controlling both
DRAT-GlnB complex formation (and DRAT activation) as well as
regulating GlnJ and, through it, DRAG association to the
chromatophore membrane. Understandably, the model here proposed
for DRAT activity regulation is based on in vitro data and, while 2OG
was shown to play a vital role, it cannot be excluded that other factors,
such as the NAD+:NADH ratio and/or the uridylylation state of GlnB,
may affect DRAT-GlnB interaction and DRAT activity in vivo.
Nevertheless, it is now proven that DRAT interaction with GlnB
greatly enhances DRAT activity. Binding of GlnB presumably
45
induces a conformational change on DRAT, increasing its affinity for
Fe protein and/or β-NAD+. The previously reported DRAT variants
K103E and N46D 207 were also assayed and further accentuate these
effects. K103E, a constitutively active DRAT variant, interacts with
GlnB in the same manner as wild-type (WT) DRAT, responding to
variations in concentration of 2OG and the ADP:ATP ratio. However,
this variant is active even in the presence of high concentrations of
ATP and 2OG, when complex formation is disrupted. This protein has
a lower Km for β-NAD+ (IV and 207) and, possibly, the conformational
change induced by the substitution of a positively-charged amino acid
(lysine) for a negatively charged residue (glutamate) increases the
affinity for the substrate(s), obviating the need for an interaction with
GlnB (IV). Conversely, the N46D variant was found to be less stable,
on account of the fact that it has a lower affinity for GlnB. This
protein also has no detectable activity in vitro, even though a very
reduced level of activity (8%) has previously been reported in vivo 207.
The substitution of residue 46 could cause a change in DRAT
conformation, inhibiting its interaction with GlnB and, therefore,
reducing affinity for its substrates (IV), once more demonstrating the
need for DRAT-GlnB interaction for efficient DRAT ADPribosylation activity.
2. Regulation of energy switch-off
As previously mentioned, R. rubrum nitrogenase can also be
modified in response to light withdrawal. This response, however, has
different characteristics depending on the nitrogen source. Even
though both glutamate-grown and N2-grown cultures will modify
nitrogenase and inhibit nitrogenase activity in response to light
withdrawal, the extent of inhibition is different between the two
conditions, with N2-grown cultures retaining some nitrogenase activity
when subjected to darkness, whereas glutamate-grown cells do not
(V). As mentioned above, it was also recently shown that PII
uridylylation patterns between glutamate- and N2-grown cultures are
different in response to light withdrawal 148. As uridylylated GlnJ is
unable to interact with AmtB1, this may also disrupt DRAG
membrane association, freeing some of this protein in the cytosol
where it can unmodify nitrogenase. As we have seen, membrane
association of DRAG is highly dependent on AmtB1 during
ammonium switch-off 144. In energy switch-off, however, AmtB1
seems to enhance but not to be required for efficient regulation of
46
DRAG membrane association in diazotrophically grown cells, thus
suggesting the presence of a yet unknown membrane partner for
DRAG 101. Simultaneously, the different modification patterns
observed for PII proteins grown diazotrophically or with glutamate
may also help explain why in AmtB1-deficient glutamate-grown R.
rubrum cultures energy switch-off is reduced to ca. 50% 144.
It was not possible to assess the effect of the modification status of
GlnB on DRAT-GlnB complex formation as the conditions required
for GlnB modification also destabilize complex formation. DRAT is
very unstable when not in complex with GlnB, forming precipitates
when present by itself at the concentrations required for such studies
and the in vitro modification reaction of GlnB will, therefore, also
lead to DRAT precipitation. Even though using a partially modified
GlnB fraction DRAT-GlnB complex formation and activity could be
observed, it cannot be excluded that complete GlnB modification may
affect DRAT activity.
In diazotrophically-grown cells the PII proteins remain modified in
response to light withdrawal and this may both destabilize DRAG
membrane association, releasing some DRAG in the cytosol, at the
same time that it could also affect DRAT activity. In this case, it is
possible that simultaneously both DRAG and DRAT activities may be
present, resulting in a fraction of the Fe protein remaining unmodified
and giving measurable (albeit much lower) nitrogenase activity (V). In
glutamate-grown cells, however, PII proteins are demodified in
response to darkness, DRAG is found only by the membrane, DRAT
is fully active and the Fe protein is completely modified, with no
measurable nitrogenase activity (V).
Addition of pyruvate to glutamate-grown cultures induces protein
synthesis. Even though it was not possible to identify the newly
synthesized protein, the outcome of pyruvate addition is the shift from
a “glutamate-response” to an “N2-response”, inducing PII protein
remodification after a period of 60 to 90 minutes. At the same time,
and responding to this shift in PII modification, GS is also reactivated.
Nitrogenase activity is regained, to the same level of N2-grown cells,
with the Fe protein becoming unmodified (V). It can, therefore, be
concluded that pyruvate addition will ultimately result in some DRAG
release from the membrane, perhaps due to GlnJ modification, and
that DRAT will become (at least partially) inactivated. Upon light
withdrawal 2OG levels do decrease (from 5.34 ± 0.11 to 2.9 ± 0.23
nmol 2OG/mg protein – unpublished) in glutamate-grown cells and
pyruvate addition allows them to recover to levels similar to those of
cells in the light (6.49 ± 0.15 nmol 2OG/mg protein – unpublished).
47
However, all of these values are still higher than that found in
diazotrophically-grown cells in the light (2.35 ± 0.34 nmol 2OG/mg
protein 93). As such, variations in the intracellular 2OG pool, even
though very important, are not the only factor regulating DRAT-GlnB
interaction and DRAT activity.
Pyruvate can be utilized by R. rubrum through different pathways
and one of them includes the enzyme pyruvate formate-lyase (Pfl).
This pathway (Figure 8), also present in other organisms, allows
production of ATP by substrate-level phosphorylation in anaerobic
conditions. In E. coli Pfl is induced upon transfer to low oxygen
tensions, especially in the presence of pyruvate and expression of Pfl
was shown to be dependent upon the FNR and ArcA/B redox sensors
208, 209
.
Figure 8 – Pyruvate utilization pathway through Pfl; Fhl – Formate hydrogenlyase;
Pta – phosphotransacetylase; Ack – Acetate kinase
R. rubrum was also shown to use this pathway when pyruvate was
supplied as the carbon source under anaerobic conditions in the dark
210
. In the case of diazotrophically-grown cells, light withdrawal
naturally inhibits nitrogenase activity, as mentioned, but a low level of
activity can still be measured immediately after transfer to the dark,
with or without addition of pyruvate. However, glutamate-grown cells
grown with malate as a carbon source cannot maintain measurable
nitrogenase activity in the dark. Pyruvate addition, as we have
discussed, induces protein synthesis and recovers nitrogenase activity
to a low level under the same conditions. This newly synthesized
protein(s) should already be present under diazotrophic conditions, so
as to maintain anaerobic dark nitrogenase activity. Although it was not
possible to assess the levels of Pfl in either condition, the enzymes in
this pathway (or others regulating their activity) are likely candidates
to be the proteins expressed upon pyruvate addition. Inhibition of this
pathway by the addition of the specific inhibitor hypophosphite 211, 212
48
not only induces Fe protein modification, inhibiting nitrogenase
activity, but it also induces demodification of the PII proteins, GS
modification (V) and a slight decrease in the intracellular 2OG pool
(from 6.49 ± 0.15 to 5.06 ± 0.32 nmol 2OG/mg protein –
unpublished). These effects can be seen in both diazotrophically- and
glutamate-grown cultures. Again, the PII modification status could
account for a higher affinity of DRAG to the membrane, as under
these conditions the AmtB1-GlnJ complex will once more be
favoured. However, considering this pathway is also important for
ATP generation, it could be that the ADP:ATP ratio, which could
increase upon light withdrawal, would also play an important role on
regulating DRAT-GlnB complex formation. Even though several
studies have addressed this phenomenon 213-215, data on ADP:ATP
ratios upon light withdrawal in diazotrophically-grown R. rubrum
cultures remains scarce and sometimes contradictory. However, it
seems likely that, upon transfer from light (with photosynthetical
generation of ATP) to dark (with fermentative ATP production), there
should be at least a transient shift in ADP:ATP ratios in the cell. This
may also affect binding of 2OG by the PII proteins which, as we have
discussed, requires the presence of ATP, thus influencing PII
interactions with their cellular targets. Furthermore, it was shown in
Rb. capsulatus that an unmodifiable GlnK variant still followed the
same pattern of association to AmtB in response to switch-off
effectors, further emphasizing that the binding of small effectors
(ADP:ATP and 2OG) to the PII proteins, rather than modification
status alone, is responsible for interaction regulation 216.
Another factor to take into consideration is that the NAD(P)+:
NAD(P)H ratios were also shown to influence nitrogenase switch-off,
with addition of NAD+ inducing transient Fe protein modification and
loss of nitrogenase activity 153 and, conversely, the addition of switchoff effectors led to the oxidation of the NAD(P) pool 152. It is not yet
understood how a change in the NAD(P) pool is sensed by the
DRAT/DRAG system but it could be that increased substrate
availability (NAD+) plays a role in inducing DRAT activity. This
could also be reflected on the redox state of the Fe protein itself, as it
was shown that DRAT has a higher affinity for the oxidized form and
DRAG prefers reduced Fe protein 133. A more oxidised pool of
NAD(P) could then coincide with more oxidized Fe protein and, thus,
stimulate DRAT activity, at the same time that DRAG activity would
be inhibited.
Although the full mechanism controlling DRAT and DRAG activity
during energy switch-off in R. rubrum is still not fully understood, it
could be that several or even all of the factors mentioned above, in
combination, affect its regulation.
49
IV. Future prospects
The study of nitrogen fixation has lead to many important advances
in recent years, with insights into enzyme structures (such as DRAG)
and regulatory schemes and many important details regarding switchoff regulation now being known. However, as it usually happens,
some of these answers only bring about new unanswered questions.
The identity of the regulatory system linking redox sensing and
differential protein expression in R. rubrum, for instance, is one of
them. As we have discussed, this system should sense, directly or
indirectly, changes in the cellular redox poise, perhaps by interaction
with quinones in the chromatophore membrane, and it would be of
great interest to identify its components and understand its regulation.
Similarly, some of the aspects regarding regulation of DRAG
membrane association still remain unresolved. Several different
strategies were employed to try to identify the membrane interacting
partner(s) for DRAG but so far this search has been unsuccessful,
even though the AmtB1-GlnJ complex has been implicated at least in
ammonium switch-off regulation. Whether such a binding partner
actually exists is also still a matter of debate although, in addition to
the factors described above, the fact that DRAG can also be found by
the membrane in nitrogen rich conditions (when the expression of
AmtB1 and GlnJ is negligible 101) gives some support to that theory.
Much is still left to be understood of the mechanism regarding
energy switch-off in R. rubrum. As this enzyme seems to be so
important for maintenance of nitrogenase activity in dark anaerobic
conditions, generating an R. rubrum strain lacking Pfl could be very
informative. Also, development of a direct, accurate and real-time in
vivo assay for estimation of the ADP:ATP ratios during energy (and
even nitrogen) switch-off is highly desirable, to establish the
importance of this factor in switch-off control. With the recent
development of new in vivo sensors, such as the one using a modified
version of a PII protein 91, this goal may now be closer at hand.
50
V. Concluding remarks
Efficient regulation of metabolic processes is vital for all living
beings. In this thesis I have approached the strategies used by R.
rubrum to adapt to different nitrogen availability conditions. As
nitrogen is such an essential element for life, nitrogen fixing bacteria
devote a large amount of their cellular resources to the maintenance
and regulation of nitrogen fixation and assimilation. We have seen
how induction of nitrogen fixation in R. rubrum represses carbon
fixation and utilization pathways (I). I have also discussed how
intracellular 2OG levels vary between nitrogen fixing and nitrogen
rich conditions and how a manipulation of the intracellular 2OG pool
will affect the co-regulation of both nitrogen and carbon fixation
pathways (II). As the 2OG concentration affects PII uridylylation, the
processes in which PII protein uridylylation plays an important role,
such as NifA activation and GS regulation, are also affected by
changes in the 2OG pool (II). Furthermore, the interconnection
between carbon and nitrogen fixation pathways suggests that there is a
sensor/transducer system, capable of responding to changes in the
cellular redox poise, as nitrogen fixation and carbon fixation are both
processes that require substantial reductive power. As the
chromatophore membrane plays a vital role in many cellular
processes, including nitrogen fixation, a Blue Native gel system using
a new class of amphiphilic detergents was developed, in combination
with LTQ-Orbitrap mass spectrometry. This system allowed
identification of many chromatophore membrane protein complex
components and showed that the Sdh complex is up regulated in
nitrogen fixing conditions (III), providing further strength to the
concept of a redox-sensing regulator. As nitrogen fixation requires
large amounts of reducing equivalents, this can also be seen as
evidence supporting the importance of the TCA cycle for nitrogenase
activity and, perhaps, also supporting the reversed electron flow
mechanism in R. rubrum as a mean to generate NADH and reoxidise
the quinone pool.
I have also described for the first time how DRAT and the PII
protein GlnB from R. rubrum form not only a complex but a complex
with measurable DRAT activity. Complex formation, and
consequently DRAT activity, is regulated by binding of the small
51
effectors ADP/ATP and 2OG, presumably to the PII protein, with
lower ADP:ATP ratios and the presence of 2OG destabilizing the
complex and inactivating DRAT (IV). As this will undoubtedly
provide a clear insight into the regulatory mechanism behind DRATGlnB complex formation, a project is also under way to obtain the
atomic structure of this complex.
While the binding of 2OG could explain regulation of DRAT activity
in nitrogen switch-off, the regulation of energy switch-off is still
unclear. Different nitrogen sources influence the response to light
withdrawal in different manners, with diazotrophically-grown cells
maintaining some activity in the dark, unlike glutamate-grown cells.
Addition of pyruvate to glutamate cultures induces protein synthesis
and allows recovery of partial nitrogenase activity in the dark. The
work presented in this thesis also shows how the activity of the
pathway starting in Pfl is vital for dark anaerobic nitrogenase activity
and that the PII proteins, nitrogenase and GS modification status
respond to pyruvate addition and Pfl pathway inhibition (V). It could
be that this pathway is already present in diazotrophically-grown cells,
providing some of the ATP needed to maintain a certain “threshold”
of the ADP:ATP ratio, which maintains PII modification and affects
DRAT/DRAG regulation. Further studies are still required in order to
fully understand the mechanisms regulating energy switch-off and the
influence of not only ADP:ATP but also of NAD(P)H:NAD(P)+ ratios
for DRAT and DRAG activity regulation.
Even though many new aspects have now come to light, the
regulation of nitrogen fixation in R. rubrum still has countless
interesting questions, waiting to be answered.
52
VI. Sumário em português
A adaptabilidade é uma das chaves do segredo das diferentes formas
de vida presentes no nosso Planeta. De entre todos os diferentes
grupos de organismos, as bactérias são, indubitavelmente, os de maior
sucesso, sendo capazes de se adaptar às mais diversas condições. São
também bactérias as responsáveis por diversos processos vitais para a
manutenção da vida na Terra, como seja a fixação de azoto. Este
elemento constitui cerca de 80% da atmosfera, sob a forma de N2
gasoso. Contudo, e apesar desta extrema abundância, a
disponibilidade deste elemento é muitas vezes limitada. O processo de
fixação de azoto, no qual o azoto atmosférico (indisponível para a
maioria dos organismos) é convertido em amónia (uma forma que
pode ser utilizada pela biosfera para síntese de moléculas vitais como
aminoácidos, DNA ou RNA, entre outros) é pois de importância vital.
A enzima responsável por este processo, nitrogenase, é extremamente
sensível ao oxigénio e, dado que a sua reacção requer quantidades
consideráveis de energia (sob a forma de ATP e equivalentes
redutores), está sujeita a variados mecanismos de regulação, tanto précomo pós-tradução. Modificação de uma subunidade da nitrogenase
inibe a reacção de fixação de azoto, permitindo a regulação da
actividade de forma instantânea.
Nesta tese são descritos os mecanismos de controlo presentes na
bactéria fotossintética e fixadora de azoto Rhodospirillum rubrum,
presente em sedimentos de rios e lagos por todo mundo. Através de
técnicas de proteómica, algumas desenvolvidas especificamente para
este trabalho, foi possível demonstrar que os processos de fixação de
azoto e fixação de carbono são co-regulados, possivelmente de
maneira a manter o equilíbrio redox no interior da célula. Para além da
regulação do proteoma, a regulação da actividade da nitrogenase por
modificação pós-tradução é também objecto de estudo e, pela primeira
vez, é demonstrado que a enzima responsável pela modificação da
nitrogenase (DRAT) é regulada através da interacção com uma
proteína pertencente à família de proteínas PII – GlnB. A formação
deste complexo, a forma activa da enzima, é regulada pela razão entre
as concentrações de ADP e ATP e 2-oxoglutarato. Estes resultados
permitem a elaboração de um modelo de regulação pós-tradução da
nitrogenase, em resposta às concentrações de amónio.
53
A enzima nitrogenase pode ser modificada tanto em resposta a um
aumento das concentrações de amónio (dado que nesse caso não é
necessário haver fixação de azoto) como por ausência de luz. A
resposta à escuridão é também descrita nesta tese e é demonstrada pela
primeira vez que a actividade da enzima piruvato formato-liase é
essencial para manutenção da actividade da nitrogenase na ausência de
luz e oxigénio.
Em conclusão, os resultados obtidos com este estudo permitem a
elaboração de um modelo de regulação do processo de fixação de
azoto em R. rubrum, aumentando o nosso conhecimento acerca deste
processo crucial para a vida na Terra.
54
VII. Acknowledgements
“How many roads must a man walk down
Before you call him a man?”
Bob Dylan, in “Blowin’ in the wind”
Dear reader, welcome to the most popular pages in the thesis!!! Please do take the
time to peruse the previous 50 or so pages though… I did my best to write an
interesting text! :o)
It is no secret to anyone that the road to those three letters (PhD) is long, tricky,
frustrating and many times can lead to “Insanityville”… but it is also immensely
rewarding, mostly for the people one meets along the way. For reasons of space, I
won’t be able to thank everyone I would like but, don’t worry, I did not forget about
you…! :o)
First and foremost, I thank my supervisors, Agneta Norén and Stefan Nordlund.
You accepted me into the “Fixer family” without even knowing me, backed me up
200%, through thick and thin. You put up with my crazy ideas and were always
there for me, with good advice, a few (needed, I know…!) “slaps in the hand” and
all the help I could have ever asked for, both in and out of the lab. Thank you for
“keeping my eyes on the ball” and helping me grow, both as a scientist and (I hope!)
as a person. None of my words will ever repay you enough… but I still had to give it
a shot! :o)
Second, to my good friend, companion and “brother-in-arms”, Dr. (ah pois!!!)
Pedro Teixeira. Tive a honra e felicidade de partilhar esta última década contigo, os
últimos anos aqui pelo Norte – aliás, não fosses tu provavelmente nunca teria sequer
ido para a Suécia... É bom ter amigos, mas melhor ainda é ter bons amigos – e
felizmente que te posso contar nesse grupo! Não sei como raio fazes para saber tanta
coisa... mas sem dúvida que mais que uma vez foi bom poder contar contigo, tanto
para saber qualquer coisa no laboratório como também quem marcou o golo
vencedor no Alguidares de Baixo F. C. vs U. D. R. Freixo de Espada à Cinta!! :oD
Aqui fica, portanto, o meu abraço, por todos os bons momentos que passámos e por
todos os que hão-de vir! Votos de sucesso em tudo, por toda a vida! E biba o F.C.P.!
55
To my co-workers, lab-mates and friends, Tomas Edgren, Wang He and Anders
Jonsson – thanks guys! Tomas, I will always admire your positive attitude towards
life and your apparently never-ending knowledge. Oh, and thanks for teaching me
about beer!!! :oD Helen – ah, what can I say? Life just isn’t the same around here
without you! Thanks for all the things you taught me, all the good advice, about
science and other things – ;o) – all the interesting experiences and, most of all, for
being a good friend. Take care of the Big Guy – and of the Small Guy/Girl too, when
he/she shows up!!! And, of course, AJ!!! Thanks for your good humour and for all
the stories you had for us. I have to say I’d never met anyone with a life quite as
remarkable as yours! I wish you all the best!
Não esquecer o resto da “Portuguese Mafia”, claro! Às nossas meninas Catarina e
Salomé, com quem tive o prazer de compartilhar tantos e tão bons momentos por
aqui e com quem formámos o “terrível” quarteto de Portugas, sempre pronto a cortar
na casaca, fazer uma jantarada ou, simplesmente, galhofar um bocado... Muito boa
sorte, não tarda muito e são vocês!!
Since we’re speaking of “Portuguese Mafia”, I’d also like to extend that “honour”
to our “adopted” members, Pilar, Beata, Changrong, Dimitra, Candan and
Erdem. What a good time we’ve had together!! I’ll miss you guys… Also, a word
of gratitude to my previous office mates Alex G., Hanna G., Hanna E.,, Kajsa and
Anna W., for the great atmosphere we had in our little corner. All the best to each
and every one of you!
A special word of thanks to my good friends “downstairs” at the JWdG lab,
especially to Mirjam K.. I am so happy to have met you, thank you so much for
being there for me! Your friendly smile is something I have learned to appreciate
and I can tell you that it’ll be sorely missed! I wish you nothing but the very best!
To my friends from “across the yard”, Anya (Прывітаньне!!), Linnea (do the fish
face again!!!) and Minttu (Hauska tavata!), thanks for all the good memories you
gave me, it has been a pleasure! Also, to everyone at Martin Högbom’s lab,
especially to Martin, whose enthusiasm for science is truly contagious, and Mike
and Charlotta, for all the help with DRAT, a big, big thank you!
To our “cousins” from Botan, especially my friend and co-author Simina and to
the “RuBisCO subunits” Irina, Nastia and Nok. Simina, I wish you and Jack all the
best in the States! Большое спасибо, Иpa и Настя, my life definitely became more
interesting after meeting you. I will never forget you! :o)
To my students, Timo Schmidt and Veronika Henriksson, for the great times we
had together and for all I learned from you. Good luck to you both!
Aproveito também para deixar um grande abraço para dois grandes amigos (e
parceiros de badminton!), Rui e Erika. Obrigado pelo vosso apoio, bons conselhos
de “quem já passou por isso” e, especialmente ao Rui, pela ENORME ajuda!
Igualmente aqui fica um beijinho para a minha amiga Nela, a quem desejo toda a
56
sorte do mundo e um valente abraço para o “xôtor” Nuno M., de quem tenho a sorte
de ser amigo, mesmo depois de todos estes anos e de toda a distância!
Likewise, a word of appreciation to those that followed my path from far away and
always had a friendly word of encouragement. Köszkönöm szépen Zsuzsa és
Zoltán! A mes très bons amis Daniel et Thérèse Ganivet, un énorme merci
beaucoup! Et on continue en français pour remercier aussi à Claire, Arezki et
Bertrand! Y para ti también, Ana, un gran besote - y ahora yo también soy
Dotor !!! Y quizás... :o)
To Bogos, for always having a friendly word for us, for always remembering the
latest football news and for all the help throughout the years, thank you very much!
Också, tack till Ann, Maria, Lotta och Lollo som gör att DBB alltid ”lever”. Ett
speciellt tack till Eddie och Håkan för alla hjälpen med diverse (icke fungerande...)
prylar!
To all of you that make DBB such a wonderful place to work in - I’ll miss you all!
Thanks also to Fundação para a Ciência e a Tecnologia for the finantial support.
Um grande abraço a toda a minha “família adoptiva” – a família Teixeira – por
todo o apoio e amizade ao longo dos anos.
Finalmente, o maior agradecimento para a minha família, por todo o apoio,
carinho, dedicação e ajuda ao longo deste longo percurso, especialmente aos meus
pais, José e Clara Selão, às minhas avós, Alcinda Selão e Maria Madalena
Marecos, que sempre se lembram do seu neto que nunca pára em casa, ao meu tio
António José Espadinha do Monte, sempre interessado em saber notícias das
minhas amigas bactérias, e ao meu irmão Pedro Selão – muito obrigado por estarem
aí por mim. Sem vocês nunca teria conseguido resistir, este livro não é só meu, é de
todos nós no “muy grande e nobre clã Selão”! ;o)
“Caminante, son tus huellas
el camino y nada más;
caminante, no hay camino,
se hace camino al andar.
[...]
Golpe a golpe, verso a verso.“
Antonio Machado, in “Cantares”
57
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