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Patrick C. Hallenbeck Editor
Modern
Topics in the
Phototrophic
Prokaryotes
Metabolism, Bioenergetics, and Omics
Modern Topics in the Phototrophic Prokaryotes
Patrick C. Hallenbeck
Editor
Modern Topics in the

Phototrophic Prokaryotes
Metabolism, Bioenergetics, and Omics
Editor
Patrick C. Hallenbeck
Department of Biology
Life Sciences Research Center
United States Air Force Academy, USA
Département de microbiologie, infectiologie et immunologie
Université de Montréal
Montréal, Québec, Canada
ISBN 978-3-319-51363-8 ISBN 978-3-319-51365-2 (eBook)

DOI 10.1007/978-3-319-51365-2
Library of Congress Control Number: 2017933463
© Springer International Publishing AG 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
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The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Contents
egulation of Nitrogen Fixation in Photosynthetic Purple

R
Nonsulfur Bacteria...........................................................................................1
Bernd Masepohl
Sulfur Metabolism in Phototrophic Bacteria................................................ 27
Christiane Dahl
iochemistry of Chlorophyll Biosynthesis
B
in Photosynthetic Prokaryotes........................................................................ 67
Yuichi Fujita and Hisanori Yamakawa
he Maintenance of Iron Homeostasis Among
T
Prokaryotic Phototrophs................................................................................. 123
Sébastien Zappa and Carl E. Bauer
Cyanobacterial Photosynthesis: The Light Reactions.................................. 163
Sascha Rexroth, Marc M. Nowaczyk, and Matthias Rögner
Photosynthesis in the Purple Bacteria............................................................ 193
Robert A. Niederman
Proteome Analysis of Phototrophic Adaptation............................................ 225
Frédéric Deschoenmaeker, Baptiste Leroy, and Ruddy Wattiez
Photosynthetic Carbon Metabolism and CO2-Concentrating
Mechanism of Cyanobacteria......................................................................... 271
Natalia A. Pronina, Elena V. Kupriyanova, and Abir U. Igamberdiev
iosynthesis of Cyanobacterial Light-Harvesting Pigments
B
and Their Assembly into Phycobiliproteins................................................... 305
Benjamin Ledermann, Marco Aras, and Nicole Frankenberg-Dinkel
Index.................................................................................................................. 341
v
Regulation of Nitrogen Fixation
in Photosynthetic Purple Nonsulfur Bacteria
Bernd Masepohl
Abstract Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed process in

which dinitrogen (N2) is reduced to ammonia (NH3), the preferred nitrogen source in
bacteria. All N2-fixing or diazotrophic bacteria have molybdenum-nitrogenases. In
addition, some diazotrophs possess one or two alternative Mo-free nitrogenases,
namely a vanadium and/or an iron-only nitrogenase, which are less efficient than
Mo-nitrogenase in terms of ATP-consumption per N2 reduced. BNF is widespread in
photosynthetic purple nonsulfur bacteria, which are capable of using light energy to
generate ATP for nitrogenase activity. This review focusses on BNF regulation in the
purple nonsulfur bacteria Rhodobacter capsulatus, Rhodopseudomonas palustris,
and Rhodospirillum rubrum. Rp. palustris is one of few diazotrophs having both
alternative nitrogenases, whereas Rb. capsulatus and Rs. rubrum have Fe-nitrogenases
but no V-nitrogenase. Purple nonsulfur bacteria regulate BNF in response to ammo-
nium, molybdenum, iron, oxygen, and light. BNF regulation involves common regu-
latory proteins including the two-component nitrogen regulatory system NtrB-NtrC,
the transcriptional activator NifA, the nitrogen-specific sigma factor RpoN, the
DraT-DraG system for posttranslational nitrogenase regulation, and at least two PII
signal transduction proteins. When ammonium is limiting, NtrB phosphorylates
NtrC, which in turn activates expression of nifA and other BNF-related genes. NifA
and its homologs VnfA and AnfA activate expression of Mo, V, and Fe-nitrogenase
genes, respectively, in concert with RpoN. DraT mediates nitrogenase switch-off by
ADP-ribosylation upon ammonium addition or light deprivation, the latter condition
causing energy depletion. DraG reactivates nitrogenase upon ammonium consump-
tion or reillumination. PII-like proteins integrate the cellular nitrogen, carbon, and
energy levels, and control activity of NtrB, NifA, DraT, and DraG. Beside these
similarities in BNF regulation, there are species-specific differences. NifA is active
as synthesized in Rb. capsulatus, but requires activation by PII in Rp. palustris and
Rs. rubrum. Reversible ADP-ribosylation is the only mechanism regulating nitroge-
nase in Rs. rubrum, whereas Rb. capsulatus and Rp. palustris have additional ADP-
ribosylation-independent mechanisms. Last but not least, molybdate directly
B. Masepohl (*)
Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und Biotechnologie,
Ruhr-Universität Bochum, 44780 Bochum, Germany
e-mail: [email protected]
© Springer International Publishing AG 2017 1

P.C. Hallenbeck (ed.), Modern Topics in the Phototrophic Prokaryotes,
DOI 10.1007/978-3-319-51365-2_1
2 B. Masepohl
represses anfA transcription and hence, Fe-nitrogenase expression in Rb. capsulatus,

whereas expression of the alternative nitrogenases in Rp. palustris and Rs. rubrum
respond to Mo-nitrogenase activity rather than to molybdate directly.
Keywords Nitrogen fixation • Nitrogenase • Rhodobacter • Rhodopseudomonas •

Rhodospirillum • Regulation
Introduction to Biological Nitrogen Fixation
Growth of all eukaryotes and most prokaryotes requires a fixed nitrogen source
like ammonium, nitrate, or amino acids. Quite a few prokaryotes, however, can
reduce the chemically inert molecular dinitrogen (N2) to ammonia (NH3) by a pro-
cess called biological nitrogen fixation (BNF). No eukaryote is capable of directly
fixing N2 but several eukaryotes like legumes and termites make indirectly use of
N2 by forming symbiotic associations with nitrogen-fixing bacteria or archaea
(Hongoh 2010; Oldroyd 2013). BNF depends on complex metalloenzymes called
nitrogenases, which catalyze the overall reaction shown in Eq. (1), and require a
theoretical minimum of 16 ATP per N2 reduced (Igarashi and Seefeldt 2003). In
addition to N2 reduction, nitrogenases produce hydrogen gas (H2) in an obligate
side reaction and, in the absence of N2, nitrogenases exclusively reduce protons to
H2. Nitrogenase-catalyzed production of H2 as a biofuel has extensively been stud-
ied in photosynthetic bacteria (Adessi et al. 2016; Heiniger et al. 2012; Huang
et al. 2010; McKinlay and Harwood 2010; Rey et al. 2007) and is discussed in
more detail elsewhere in this book series.
N 2 + 8 e − + 8 H + + 16 ATP → 2 NH3 + H 2 + 16 ( ADP + Pi ) (1)
All N2-fixing (diazotrophic) bacteria and archaea possess a molybdenum-

dependent nitrogenase containing the catalytic iron-molybdenum cofactor,
FeMoco (Zhang and Gladyshev 2008). In addition to Mo-nitrogenase, some
diazotrophs synthesize alternative Mo-free nitrogenases containing the iron-
vanadium cofactor, FeVco, or the iron-only cofactor, FeFeco (Dos Santos et al.
2012; McGlynn et al. 2013). The three nitrogenases are encoded by distinct gene
sets, namely nifHDK (Mo-nitrogenase), vnfHDGK (V-nitrogenase), and anfH-
DGK (Fe-nitrogenase). Beside the structural nitrogenase genes, diazotrophs have
numerous genes involved in cofactor biosynthesis, electron supply, and regula-
tion (see below).
In addition to N2 and protons, nitrogenases reduce the artificial substrate acety-
lene (C2H2). Mo-nitrogenases reduce acetylene to ethylene (C2H4), whereas alterna-
tive nitrogenases reduce acetylene to ethylene and in part, to ethane (C2H6). In the
laboratory, gas chromatography-based acetylene reduction assays have been estab-
lished to quantify nitrogenase activity and to detect activity of alternative nitroge-
nases (Dilworth et al. 1988).
Regulation of Nitrogen Fixation in Photosynthetic Purple Nonsulfur Bacteria 3
Mo, V, and Fe-nitrogenases consist of two components each, the catalytic dini-
trogenases and the dinitrogenase reductases, the latter serving as the ultimate elec-
tron donors to their respective dinitrogenases (Curatti and Rubio 2014; Hu and
Ribbe 2016). The three dinitrogenase reductases are collectively called Fe-proteins
(homodimers of NifH, VnfH, and AnfH), all of which coordinate one [4Fe-4S]
cluster involved in electron transfer. The Mo, V, and Fe-dinitrogenases are called
MoFe-protein (heterotetramer of NifDK containing two FeMoco), VFe-protein
(heterohexamer of VnfDGK containing two FeVco), and FeFe-protein (heterohex-
amer of AnfDGK containing two FeFeco), respectively. In addition to the catalytic
cofactors, the dinitrogenases contain two P-clusters (see below) involved in electron
transfer from the Fe-proteins to the catalytic cofactors.
Biosynthesis of the Mo-nitrogenase cofactors ([4Fe-4S] cluster, P-cluster,
and FeMoco) is complex and requires several nif gene products including NifU,
NifS, NifB, NifV, NifE, NifN, NifH, NifD, and NifK as shown for Klebsiella
pneumoniae and Azotobacter vinelandii (Curatti and Rubio 2014; Hu and
Ribbe 2016; and the references therein). Briefly, NifU and NifS function as the
scaffold protein and sulfur donor, respectively, for biosynthesis of [4Fe-4S]
clusters, which are either inserted into apo-NifH or serve as building blocks for
P-cluster and FeMoco formation. The P-cluster is formed in situ on the apo-
NifDK protein, whereas the FeMoco is synthesized ex situ prior to insertion
into the apo-NifDK protein. P-cluster biosynthesis starts with the transfer of
two [4Fe-4S] clusters to the apo-NifDK protein followed by NifH-mediated
reductive coupling to form the [8Fe-7S] or P-cluster. FeMoco biosynthesis
starts with the coupling of two [4Fe-4S] clusters on NifB involving S-adenosyl-
methionine-dependent carbon (C) insertion to form an [8Fe-9S-C] cluster. This
cluster is further processed on the NifEN scaffold by insertion of Mo and
homocitrate (the product of homocitrate synthase, NifV) resulting in the
[Mo-7Fe-9S-C-homocitrate] cluster or FeMoco, which is finally inserted into
the apo-NifDK protein.
NifU, NifS, NifB, and NifV are required for activity of Mo, V, and
Fe-nitrogenases in A. vinelandii indicating that the biosynthetic pathways of
FeMoco, FeVco, and FeFeco overlap to a certain extent (Drummond et al. 1996;
Kennedy and Dean 1992). Formation of FeVco involves the Vnf-specific NifEN
homolog, VnfEN, instead of NifEN (Hu and Ribbe 2016; and the references
therein). Possibly, the last steps of FeFeco biosynthesis occur in situ on the
AnfDGK protein, since no NifEN homolog is required for Fe-nitrogenase activ-
ity (Schüddekopf et al. 1993).
Diazotrophs regulate BNF in response to several environmental factors including
ammonium, molybdenum, iron, oxygen, and in case of photosynthetic bacteria,
light. Since BNF is a highly energy-demanding process, diazotrophs typically
induce nitrogenase expression only when ammonium, the product of BNF, is limit-
ing. Mo-nitrogenase is more efficient than the alternative nitrogenases in terms of
consumption of ATP and reductant per N2 reduced (Hu et al. 2012; Schneider et al.
1997) and hence, expression of alternative nitrogenases is typically repressed as
long as Mo-nitrogenase is active. Most bacteria possess modABC genes encoding
4 B. Masepohl
high-affinity ABC transporters, which support uptake of molybdate, the only bio-
available form of molybdenum, under Mo-limiting conditions (Zhang and Gladyshev
2008; Zhang and Gladyshev 2010). All three nitrogenases are irreversibly damaged
by oxygen (Blanchard and Hales1996; Chisnell et al. 1988; Gollan et al. 1993) and
diazotrophs have evolved different strategies to cope with this problem.
Diazotrophic and non-diazotrophic bacteria utilize similar proteins to sense the
cellular nitrogen status and to control nitrogen assimilation. Among these proteins
are the bifunctional uridylyltransferase/uridylyl-removing enzyme GlnD, the PII sig-
nal transduction proteins GlnB and GlnK, the two-component regulatory system
NtrB-NtrC, and the ammonium transporter AmtB, which are best characterized in
the non-diazotrophic enterobacterium Escherichia coli (van Heeswijk et al. 2013;
and the references therein).
Briefly, GlnD senses the cellular nitrogen status through the glutamine level
(Jiang et al. 1998a). Under low glutamine levels (N-limiting conditions), GlnD
modifies GlnB and GlnK by uridylylation of conserved tyrosine residues within
their T-loops. Under high glutamine levels (N-replete conditions), GlnD cata-
lyzes the reverse reaction by hydrolyzing GlnB-UMP and GlnK-UMP. Trimeric
PII proteins can be fully uridylylated (PII-UMP3), partially uridylylated (PII-
UMP2 or PII-UMP1), or completely unmodified (PII). PII proteins directly sense
the cellular carbon and energy status by binding 2-oxoglutarate (2OG) and ATP/
ADP, respectively (Radchenko et al. 2013). 2OG joins nitrogen and carbon
metabolism as it serves as the carbon skeleton for ammonium assimilation by
the GS-GOGAT (glutamine synthetase–glutamate synthase) pathway. Taken
together, PII proteins integrate the cellular nitrogen (glutamine), carbon (2OG),
and energy (ATP/ADP) levels, and transduce these signals to target proteins by
physical interaction.
Under N-limiting conditions, the response regulator NtrC is phosphorylated by
its cognate sensor kinase NtrB (Jiang et al. 1998b). In turn, NtrC-P activates tran-
scription of glnA encoding glutamine synthetase, the glnK-amtB operon, and
genes required for generation of ammonia from “poor” nitrogen sources like
amino acids. Under N-replete conditions, unmodified GlnB forms a complex with
NtrB to stimulate dephosphorylation and hence, inactivation of NtrC. In parallel,
unmodified GlnK forms a complex with AmtB thereby inhibiting ammonium
uptake under N-replete conditions.
This review deals with the regulation of nitrogen fixation in photosynthetic purple
nonsulfur bacteria, which are capable of using light energy to generate the ATP
required for nitrogenase activity. Purple nonsulfur bacteria are known for their
extreme metabolic versatility enabling growth under photoautotrophic, photohetero-
trophic, chemoautotrophic, and chemoheterotrophic conditions (Madigan et al.
1984). BNF is widespread in purple nonsulfur bacteria and has been extensively
studied in Rhodobacter capsulatus, Rhodopseudomonas palustris, and Rhodospirillum
rubrum, whose complete genome sequences have been determined (Larimer et al.
2004; Madigan et al. 1984; Munk et al. 2011; Strnad et al. 2010). In addition to
Mo-nitrogenase, Rb. capsulatus and Rs. rubrum synthesize Fe-nitrogenases (Davis
et al. 1996; Lehman and Roberts 1991; Schneider et al. 1991; Schneider et al. 1997),
whereas Rp. palustris is one of the few diazotrophs synthesizing Mo, V, and
Fe-nitrogenases (Oda et al., 2005).
rganization of Nitrogen Fixation Genes in Purple Nonsulfur

O
Bacteria
All diazotrophs including Rb. capsulatus, Rp. palustris, and Rs. rubrum con-
tain a common set of nitrogen fixation genes, namely the structural genes of
Mo-nitrogenase (nifH, nifD, and nifK) and genes involved in [4Fe-4S] cluster,
P-cluster, and FeMoco biosynthesis (nifU, nifS, nifB, nifV, nifE, and nifN)
(Curatti and Rubio 2014; Hu and Ribbe 2016; Larimer et al. 2004;MacKellar
et al. 2016; Masepohl and Klipp 1996; Munk et al. 2011; Oda et al. 2005;
Strnad et al. 2010; Wang et al. 2013). These common nif genes cluster with
species-specific nif genes involved in regulation, electron transport to nitroge-
nase, and genes of unknown function (Fig. 1). Expression of common and spe-
cies-specific nif genes requires the central transcriptional activator NifA, which
enhances transcription by RNA polymerase containing the nitrogen-specific
sigma factor RpoN (also called NtrA or σ54) as is the case in other proteobacte-
rial diazotrophs (see below). NifA proteins consist of an N-terminal GAF
domain involved in the response to the cellular nitrogen status, a central AAA
domain involved in the interaction with RNA polymerase and ATP hydrolysis,
and a C-terminal HTH (helix-turn-helix) domain involved in binding to pro-
moter DNA (Fischer 1994). Noteworthy, Rb. capsulatus synthesizes two struc-
turally and functionally highly similar NifA proteins: NifA1 and NifA2
(Masepohl et al. 1988; Paschen et al. 2001).
Electron transport to nitrogenase in Rb. capsulatus involves the rnfABCDGEH
genes (Jeong and Jouanneau 2000; Jouanneau et al. 1998; Kumagai et al. 1997;
Schmehl et al. 1993), which are lacking in Rs. rubrum and Rp. palustris. Instead, the
latter two strains contain the fixABCD genes, whose products form the major electron
transport pathway in Rs. rubrum (Edgren and Nordlund 2004) and possibly also in
Rp. palustris (Huang et al. 2010).
Many diazotrophs including Rb. capsulatus and Rp. palustris have iscN-nifUSVW
operons, whereas Rs. rubrum lacks an nifS gene at the corresponding position
between nifU and nifV. However, Rs. rubrum contains three nifS-like genes else-
where in the chromosome, one of which possibly serves as a sulfur donor for biosyn-
thesis of iron-sulfur clusters under N2-fixing conditions.
The structural genes of Fe-nitrogenase anfHDGK and the Fe-nitrogenase-
associated genes anfOR form conserved operons in Rh. capsulatus, Rp. palustris,
and Rs. rubrum (Fig. 2) (Larimer et al. 2004; Munk et al. 2011; Oda et al. 2005;
Schüddekopf et al. 1993; Strnad et al. 2010). Expression of these anf operons is acti-
vated by AnfA, an NifA-like regulator (Kutsche et al. 1996; Schüddekopf et al.
1993). Activation of the V-nitrogenase-related genes vnfH, vnfDGK, and vnfENX in
Rp. palustris depends on VnfA, another NifA-like activator. Like NifA, AnfA and
VnfA act in concert with the sigma factor RpoN.
6
Rhodobacter capsulatus
Rhodopseudomonas palustris
Rhodospirillum rubrum
Regulation Nitrogenase Cofactor synthesis Electron transport Unknown function
Fig. 1 Organization of Mo-nitrogenase-related genes. Genetic maps are based on the genome sequences of Rh. capsulatus SB 1003 (Strnad et al. 2010), Rp.
palustris CGA009 (Larimer et al. 2004), and Rs. rubrum S1 (Munk et al. 2011). Bent arrows in red or black mark possible NtrC and RpoN recognition
B. Masepohl
sequences, respectively
Rhodobacter capsulatus
Rhodopseudomonas palustris
Regulation
Rhodospirillum rubrum
Nitrogenase
Cofactor synthesis
Unknown function
Fig. 2 Organization of Fe and V-nitrogenase-related genes. Genetic maps are based on the genome
sequences of Rh. capsulatus SB 1003 (Strnad et al. 2010), Rp. palustris CGA009 (Larimer et al.
2004), and Rs. rubrum S1 (Munk et al. 2011). Bent arrows in red or black mark possible NtrC and
RpoN recognition sequences, respectively
ascade Activation of Nitrogen Fixation in Rhodobacter

C
capsulatus
Rb. capsulatus is capable of growing with many different nitrogen sources including
ammonium, urea, most amino acids, and N2 (Hillmer and Gest 1977; Masepohl et al.
2001). Expression of urease and N2 fixation genes strictly depends on NtrC (Hübner
et al. 1991; Kranz and Haselkorn 1985; Kutsche et al. 1996; Masepohl et al. 2001).
As described above for E. coli, Rb. capsulatus NtrC is phosphorylated, and thus acti-
vated, by NtrB under ammonium-limiting conditions (Cullen et al. 1996). In contrast
to NtrC from E. coli and other bacteria, which require the nitrogen-specific sigma
factor RpoN to activate transcription of their target genes, Rb. capsulatus NtrC acti-
vates gene expression in concert with the housekeeping sigma factor RpoD (Bowman
and Kranz 1998; Foster-Hartnett et al. 1994).
Upon phosphorylation, Rb. capsulatus NtrC activates transcription of nifA1,
nifA2, mopA-modABC, and anfA (Fig. 3). Activation involves binding of NtrC to
sequences similar to the Rb. capsulatus NtrC binding site consensus CGCC–N9–
GGC–N4–14–CGCC–N9–GGC (Foster-Hartnett and Kranz 1994; Kutsche et al.
1996). NifA1 and NifA2 differ only in their very N-terminal amino acid residues,
and consequently, can functionally substitute for each other in transcriptional acti-
vation of Mo-nitrogenase genes (Masepohl et al. 1988; Paschen et al. 2001).
Expression of Fe-nitrogenase genes is activated by AnfA (Kutsche et al., 1996).
Transcriptional activation by NifA1, NifA2, and AnfA depends on RpoN as is the
8 B. Masepohl
Growth with NH4+ NtrC inact ive
N2-f ixing conditions NtrC P
D nifA1 D nifA2 D mopA modABC D anfA
Mo uptake
NifA1 NifA2 MopA AnfA
Mo-replete conditions
N nifHDK N nifHDK
Mo-nitrogenase Fe-nitrogenase
N2 NH3 N2 NH3
Fig. 3 Cascade regulation of nitrogen fixation in Rh. capsulatus. During growth with ammonium,
NtrC is inactive, but is activated by phosphorylation upon ammonium consumption. NtrC-P activates
RpoD-dependent promoters (boxed D), whereas NifA1, NifA2, and AnfA activate RpoN-dependent
promoters (boxed N). MopA represses transcription of the mopA-modABC and anfA genes under
Mo-replete conditions. For clarity, the second Mo-responsive regulator, MopB, is not shown
case in other proteobacterial diazotrophs (Hübner et al. 1991; Schüddekopf et al.

1993). The Rb. capsulatus rpoN gene forms part of the nifU2-rpoN operon, whose
expression is activated by NifA1, NifA2, and presumably also by AnfA (Cullen
et al. 1994; Preker et al. 1992). A weak primary NtrC-independent promoter located
in the nifU2-rpoN intergenic region drives initial expression of rpoN, while a sec-
ondary promoter upstream of nifU2 is required to increase rpoN expression under
N2-fixing conditions (Cullen et al. 1994).
All Rb. capsulatus nif promoters including the nifU2 promoter as well as the
anfH promoter contain sequences highly similar to the RpoN binding site consensus
CTGC–N8–TTGC typically located at position −24/−12 relative to the transcription
start site (Fig. 1) (Morett and Buck 1989; Schmehl et al. 1993). The nif promoters
are preceded by sequences similar to the NifA binding site consensus TGT–N10–
ACA (Morett and Buck 1988). As expected for an AnfA-dependent promoter, the
anfH promoter lacks an NifA binding site; however, the AnfA binding site has not
yet been identified.
mmonium Inhibition of Nitrogen Fixation in Rhodobacter

A
capsulatus
The levels of Rb. capsulatus NtrC remain constant under N-limiting and N-replete
conditions, but NtrC activity clearly responds to the cellular nitrogen status (Cullen
et al. 1998). Ammonium keeps NtrC in its dephosphorylated inactive state, thus pre-
venting expression of the nifA1, nifA2, and anfA genes, and consequently, all the
other nitrogen fixation genes (Foster-Hartnett and Kranz 1992; Preker et al. 1992).
Ammonium addition to an N2-fixing Rb. capsulatus culture causes three effects,
namely (1) inactivation of NtrC-P by dephosphorylation, (2) inhibition of NifA1,
NifA2, and AnfA activity, and (3) “switch-off” of Mo and Fe-nitrogenases
(Drepper et al. 2003; Hallenbeck 1992; Hallenbeck et al. 1982; Jouanneau et al.
1983; Masepohl et al. 1993; Paschen et al. 2001; Pierrard et al. 1993a, b;
Schüddekopf et al. 1993).
Ammonium-induced inactivation of NtrC prevents further expression of nifA1,
nifA2, and anfA. A strain lacking GlnB expresses nifA1 (and probably also nifA2 and
anfA) even in the presence of ammonium (Drepper et al. 2003). NtrB specifically
interacts with GlnB but not with GlnK (Pawlowski et al. 2003) suggesting that inac-
tivation of Rb. capsulatus NtrC is catalyzed by an NtrB-GlnB complex exhibiting
phosphatase activity as described above for E. coli.
Ammonium inhibition of NifA1, NifA2, and AnfA activity prevents further
expression of all the other nitrogen fixation genes (Paschen et al. 2001; Schüddekopf
et al. 1993). Either GlnB or GlnK is sufficient to inhibit NifA1 and NifA2, whereas
a strain lacking both PII signal transduction proteins no longer inhibits activity of the
NifA regulators (Drepper et al. 2003). Both NifA proteins interact with GlnB and
GlnK (Pawlowski et al. 2003) suggesting that NifA inhibition is mediated by physi-
cal contact with the PII proteins. The strain lacking both PII proteins still expresses
Mo-nitrogenase (Drepper et al. 2003) indicating that the Rb. capsulatus NifA pro-
teins are active as synthesized and do not require activation by PII as is the case in
Rp. palustris and Rs. rubrum (Heiniger et al. 2012; Rey et al. 2007; Zhang et al.
2000, 2004; Zhou et al. 2008; Zhu et al. 2006). In contrast to PII-mediated NifA
inhibition in Rb. capsulatus, AnfA inhibition is not relieved in the strain lacking both
PII proteins indicating that ammonium inhibition of NifA and AnfA involves differ-
ent mechanisms (Drepper et al. 2003).
Ammonium addition to an N2-grown culture rapidly represses activity of Mo and
Fe-nitrogenases, an effect immediately reversed upon ammonium consumption
(Hallenbeck 1992; Hallenbeck et al. 1982; Jouanneau et al. 1983; Masepohl et al.
1993; Pierrard et al. 1993a). In Rb. capsulatus, nitrogenase “switch-off” is caused by
at least two mechanisms, one blocking activity of the Fe-proteins, NifH and AnfH,
by ADP-ribosylation, and another possibly blocking the ATP or the electron supply
to nitrogenase (Förster et al. 1999; Pierrard et al. 1993a, b). Evidence for the second
mechanism comes from the observation that Rb. capsulatus strains expressing mutant
NifH proteins, which are no longer ADP-ribosylated, as well as a draTG mutant
strain still exhibit ammonium-induced nitrogenase switch-off (Förster et al. 1999;
10 B. Masepohl
Pierrard et al. 1993a; Yakunin and Hallenbeck 1998b). Ammonium-induced nitroge-

nase switch-off independent of ADP-ribosylation has been reported in many other
diazotrophs, but the underlying mechanisms are unknown (Huergo et al. 2012; and
the references therein). In Rb. capsulatus, a further nitrogenase switch-off mecha-
nism called “magnitude response” reflects the amount of added ammonium (Yakunin
and Hallenbeck 1998b; Yakunin et al. 1999).
ADP-ribosylation is catalyzed by DraT (dinitrogenase reductase ADP-ribosyl
transferase), whereas DraG (dinitrogenase reductase-activating glycohydrolase)
mediates the reverse reaction (Huergo et al. 2012; Nordlund and Högbom 2013; and
the references therein). ADP-ribosylation at arginine residue 101 of one subunit of
the Rb. capsulatus NifH homodimer is sufficient to prevent electron transfer to the
MoFe protein and consequently, N2 reduction by Mo-nitrogenase (Jouanneau et al.
1989). Proper regulation of nitrogenase modification and switch-off requires GlnB,
GlnK, and AmtB, and disruption of the amtB gene abolishes ADP-ribosylation and
switch-off (Drepper et al. 2003; Tremblay et al. 2007; Tremblay and Hallenbeck
2008; Yakunin and Hallenbeck 2002).The amtB strain exhibits wild-type growth
properties with ammonium as sole nitrogen source indicating that AmtB is dispens-
able for ammonium uptake, but primarily serves as an ammonium sensor for
ammonium-induced switch-off of nitrogenase (Tremblay and Hallenbeck 2009).
Figure 4 shows a model of DraTG-mediated ammonium regulation of
Mo-nitrogenase and possibly also of Fe-nitrogenase in Rb. capsulatus. The mecha-
nisms of DraT activation and DraG inactivation can be summarized as follows: (1)
Under N2-fixing conditions, both PII proteins are uridylylated, but upon ammonium
addition, GlnK-UMP and GlnB-UMP are deuridylylated. (2) Next, DraG is inacti-
vated by membrane sequestration as a ternary DraG-GlnK-AmtB complex and
DraT is activated by complex formation with GlnB. In turn, the GlnB-DraT com-
plex mediates ADP-ribosylation of the Fe-protein. (3) Upon ammonium consump-
tion, DraG is released from the membrane and reactivates the Fe-protein by
removing the ADP-ribose moiety.
mmonium Regulation of Nitrogen Fixation

A
in Rhodopseudomonas palustris
Expression and activity of Mo-nitrogenase in Rp. palustris is regulated at three lev-

els, namely (1) control of nifA transcription, (2) control of NifA activity, and (3)
switch-off control of Mo-nitrogenase as is the case for Rb. capsulatus (Heiniger et al.
2012; Rey et al. 2007). Ammonium regulation at all three levels involves PII proteins
in both diazotrophs. In contrast to Rb. capsulatus, which has two PII genes, glnB and
glnK, Rp. palustris has three PII genes forming part of the glnB-glnA and the glnK1-
amtB1-glnK2-amtB2 clusters (Connelly et al. 2006). GlnB, GlnK1, and GlnK2
undergo uridylylation under ammonium-starved (N2-fixing) conditions, but are
deuridylylated under ammonium-replete conditions. Under N2-fixing conditions,
NtrC activates transcription of the nifA gene (level 1). Only after binding to GlnB,
N2-f ixing conditions NH4+switch-of f NH4+consumed
AmtB AmtB AmtB
GlnK
inactive DraG
GlnK UMP GlnK GlnK UMP
GlnB UMP GlnB GlnB UMP
DraG DraT inactive

GlnB
Fe-protein DraT Fe-protein DraG Fe-protein
NAD+ ADP-ribose
e– e–
MoFe-protein MoFe-protein MoFe-protein
inactive
N2 NH3 N2 NH3
Fig. 4 Model of ammonium-responsive nitrogenase regulation in Rb. capsulatus. Upon ammo-

nium addition to a nitrogen-fixing culture, GlnK-UMP and GlnB-UMP are deuridylylated. In turn,
DraT is activated by GlnB, while DraG is inactivated by GlnK-mediated membrane sequestration.
DraT-mediated ADP-ribosylation of the Fe-protein prevents electron (e−) transfer to the MoFe-
protein. Upon ammonium consumption, DraG is released from the membrane and reactivates the
Fe-protein by removing the ADP-ribose moiety
Rp. palustris NifA is capable of activating Mo-nitrogenase gene expression (level 2).
Upon ammonium addition to an N2-grown culture, GlnK2 and DraT2 form a com-
plex to inactivate Mo-nitrogenase by ADP-ribosylation. In addition, Rp. palustris
DraT2 possibly regulates electron transfer to nitrogenase as discussed for Rb. capsu-
latus DraT (Förster et al. 1999; Heiniger et al. 2012; Pierrard et al. 1993a, b). Like
Mo-nitrogenase, the V and Fe-nitrogenases in Rp. palustris are modified upon
ammonium addition (Heiniger and Harwood 2015).
Rp. palustris strains synthesizing mutant NifA* proteins with single amino acid
substitutions or small deletions in the Q-linker constitutively express nitrogenase and
produce H2 even in the presence of ammonium (Heiniger et al. 2012; Rey et al. 2007).
The Q-linker is located between the nitrogen-responsive GAF domain and the RNA
polymerase-binding AAA domain (Fischer 1994). Three observations explain, how
the nifA* mutants bypass the elaborated regulatory cascade otherwise limiting N2 fixa-
tion to ammonium-starved conditions in the wild-type. First, Rp. palustris synthesizes
low amounts of NifA independent of NtrC activation. Second, mutant NifA* proteins
do not require activation by GlnB and thus, appear to be more active than wild-type
NifA proteins. Consequently, NifA* strains overexpress Mo-nitrogenase explaining at
least in part resistance against DraT2-mediated nitrogenase switch-off. Third, DraT2
12 B. Masepohl
activity requires GlnK2, whose expression depends on NtrC, which is synthesized

only at low levels in the presence of ammonium (Conlan et al. 2005). NtrC activates
transcription of the ntrC gene in Rp. palustris (Conlan et al. 2005), whereas NtrC is
constitutively synthesized Rb. capsulatus (Cullen et al. 1998).
mmonium Regulation of Nitrogen Fixation in Rhodospirillum

A
rubrum
Transcription of nifA completely or for the most part depends on NtrC in Rb. capsu-
latus and Rp. palustris, respectively (Foster-Hartnett and Kranz 1992; Heiniger et al.
2012; Hübner et al. 1993; Preker et al. 1992; Rey et al. 2007), whereas NtrC appears
to be dispensable for nifA expression in Rs. rubrum (Zhang et al. 1995). However, the
Rs. rubrum nifA gene is preceded by a possible NtrC binding site (Fig. 1) suggesting
that NtrC contributes to nifA expression. Disruption of ntrC impairs nitrogenase
switch-off in Rs. rubrum, likely because NtrC is required for maximal glnBA expres-
sion, and GlnB is essential for DraT activation (Cheng et al. 1999; Zhang et al. 1995).
Rs. rubrum NifA is synthesized in an inactive form, which requires activation
by GlnB as is the case in Rp. palustris (Zhang et al. 2000, 2001, 2004). Neither
of the other two PII proteins synthesized by Rs. rubrum, GlnK and GlnJ, can
substitute for GlnB in NifA activation. GlnD is essential for NifA activation indi-
cating that only GlnB-UMP but not its unmodified form, GlnB, is capable of
activating NifA (Zhang et al. 2005). GlnB* variants mediating NifA activity in a
strain lacking GlnD contain single amino acid substitutions in the T-loop appar-
ently mimicking the uridylylated form of GlnB (Zhang et al. 2004; Zhu et al.
2006). NifA* variants no longer requiring activation by GlnB-UMP contain
amino acid substitutions in the N-terminal GAF domain, which is involved in
interaction between wild-type NifA and GlnB-UMP (Fischer 1994; Zhou et al.
2008). Nitrogenase activity is still switched-off by ammonium in Rs. rubrum
nifA* strains, whereas ammonium switch-off is mostly relieved in Rp. palustris
nifA* strains as described above (Heiniger et al. 2012; Rey et al. 2007; Zhou
et al. 2008). Rs. rubrum nifA* strains lacking DraT, however, exhibit high nitro-
genase activity in the presence of ammonium.
DraT-mediated ADP-ribosylation appears to be the only mechanism controlling
nitrogenase activity in Rs. rubrum (Zhang et al. 1996). In contrast, nitrogenase activ-
ity is controlled by two mechanisms, one DraT-dependent and another DraT-
independent mechanism, in many other diazotrophs including Azoarcus sp. strain
BH72, Azospirillum brasilense, Herbaspirillum seropedicae, and Rb. capsulatus
(Förster et al. 1999; Fu and Burris 1989; Huergo et al. 2012; Oetjen and
Reinhold-Hurek 2009; Pierrard et al. 1993a, b; Yakunin and Hallenbeck 1998b;
Zhang et al. 1996).
Rs. rubrum has three PII genes forming part of the glnB-glnA, glnJ-amtB1, and
glnK-amtB2 operons (Munk et al. 2011). Upon ammonium addition to an N2-grown
culture, DraT is activated by interaction with unmodified GlnB, and DraG is inacti-
vated by membrane sequestration involving AmtB1, unmodified GlnJ, and possibly

an unknown membrane protein (Nordlund and Högbom 2013; Teixeira et al. 2008;
Wang et al. 2005; Wolfe et al. 2007; Zhang et al. 2006).
Darkness Regulation of Nitrogenase
Like ammonium addition, light deprivation causes nitrogenase switch-off in photo-

synthetic bacteria (Huergo et al. 2012; Nordlund and Högbom 2013; Pierrard et al.
1993b; Selao et al. 2011; Yakunin and Hallenbeck 2002; Zhang et al. 1995, 2001,
2006). In Rs. rubrum, ammonium and darkness-induced nitrogenase regulation by
reversible ADP-ribosylation involve the same proteins, namely DraT, DraG, GlnB,
GlnJ, and AmtB1, but the signaling mechanisms transducing the cellular nitrogen
and energy levels differ (Teixeira et al. 2010; Zhang et al. 2001, 2006). While ammo-
nium addition to an N2-grown culture causes a big increase in the cellular glutamine
concentration leading to GlnD-mediated deuridylylation of GlnB-UMP and GlnJ-
UMP, light deprivation does not affect the glutamine pool or induce PII demodifica-
tion on a big scale (Li et al. 1987; Teixeira et al. 2010). Full uridylylation of trimeric
PII prevents “plug-in” interaction with AmtB, whereas partially uridylylated PII pro-
teins form a complex with AmtB in A. brasilense (Rodrigues et al. 2011). Hence,
DraG inactivation in Rs. rubrum may either be achieved by GlnJ-independent mem-
brane sequestration or involve complex formation between partially deuridylylated
GlnJ and AmtB1 (Huergo et al. 2012; Nordlund and Högbom 2013).
Iron Regulation of Electron Transport to Nitrogenase
Rb. capsulatus utilizes two parallel acting electron transport pathways to nitroge-
nase, the RnfABCDGEH-FdxN and the NifJ-NifF pathway, in which the ferredoxin
FdxN and the flavodoxin NifF act as the ultimate electron donors to NifH and pos-
sibly also to AnfH (Gennaro et al. 1996; Hallenbeck and Gennaro 1998; Jeong and
Jouanneau 2000; Jouanneau et al. 1998; Kumagai et al. 1997; Schmehl et al. 1993;
Yakunin et al. 1993; Yakunin and Hallenbeck 1998a). The Rnf proteins form an
energy-coupling NADH oxidoreductase complex that catalyzes the reduction of
FdxN. The NifJ protein is a pyruvate-flavodoxin oxidoreductase mediating electron
transfer from pyruvate to NifF. In contrast to the situation in Rb. capsulatus, the
NifJ-NifF pathway constitutes the sole electron transport pathway to nitrogenase in
K. pneumoniae (Hill and Kavanagh 1980; Shah et al. 1983).
Unlike the rnf and fdxN genes, the Rb. capsulatus nifF and nifJ genes are not con-
tained in the major nif clusters (Fig. 1). However, the nifF gene belongs to the NifA
regulon and accordingly, nifF is specifically expressed under N2-fixing conditions as is
the case for the rnf and fdxN genes (Gennaro et al. 1996; Schmehl et al. 1993). In con-
trast to nifF, the nifJ gene is expressed under ammonium-replete conditions and its
14 B. Masepohl
expression increases only slightly under N2-fixing conditions indicating that NifJ func-
tion is not restricted to N2 fixation (Yakunin and Hallenbeck 1998a). The NifJ-NifF
pathway contributes significantly to electron transfer to nitrogenase under iron-replete
conditions, but is essential under iron-limiting conditions (Gennaro et al. 1996; Yakunin
et al. 1993; Yakunin and Hallenbeck 1998a). Accordingly, nifF expression and NifF
accumulation is higher under iron-deficient than under iron-sufficient conditions, while
rnf transcription and Rnf accumulation decreases upon iron limitation (Jouanneau et al.
1998). Apparently, Rb. capsulatus copes with iron limitation by replacing the iron-
containing ferredoxin FdxN by the Fe-free flavodoxin NifF, but the iron-responsive
mechanisms controlling nifF and rnf expression remain unknown to date.
Rs. rubrum lacks rnfABCDGEH genes but instead has fixABCX genes (Fig. 1),
whose products form the major electron transport pathway to nitrogenase in this
diazotroph (Edgren and Nordlund 2004). In addition, Rs. rubrum has an nifJ-like
gene encoding a pyruvate-ferredoxin oxidoreductase (Edgren and Nordlund 2006).
In both the FixABCX and the NifJ pathways ferredoxin N (encoded by the fdxN gene
located downstream of nifB; Fig. 1) is the ultimate electron donor to nitrogenase
(Edgren and Nordlund 2005, 2006). Like Rs. rubrum, Rp. palustris lacks
rnfABCDGEH genes but has fixABCX genes (Fig. 1), which are essential for diazo-
trophic growth (Huang et al. 2010) suggesting that electron transport to nitrogenase
in Rp. palustris involves a similar mechanism as in Rs. rubrum.
Regulation of Molybdate Uptake and Alternative Nitrogenases
Most bacteria synthesize high-affinity molybdate transporters (modABC-encoded)

suggesting that they have to cope at least temporarily with Mo limitation (Zhang and
Gladyshev 2008). Under Mo-replete conditions, E. coli represses modABC transcrip-
tion by the molybdate-responsive one-component regulator ModE, thus limiting
expression of the Mo uptake system to Mo-limiting conditions. ModE binds a palin-
dromic sequence called Mo-box overlapping the modA transcription start site thereby
preventing binding of RNA polymerase (Studholme and Pau 2003).
Rb. capsulatus has two modE homologs, mopA and mopB, belonging to diver-
gently transcribed operons, mopA-modABCD and mopB (Fig. 1). Upon molybdate-
binding MopA and MopB repress transcription of the mopA-modABCD and anfA
genes by binding the Mo-boxes overlapping the transcription start sites of mopA and
anfA (Fig. 5) (Kutsche et al. 1996; Müller et al. 2010; Wiethaus et al. 2006). Either
MopA or MopB is sufficient to repress transcription from the mopA and anfA pro-
moters. Beside its role as a repressor, MopA acts as a transcriptional activator of the
mop gene encoding a molybdate-binding hexameric protein (Wiethaus et al. 2009).
In contrast to the mopA and anfA Mo-boxes, which overlap the transcription start
sites, the mop Mo-box is located at some distance upstream of the transcription start
site as expected for an enhancer binding site. In line with the proposed role of the
Mop protein in Mo storage, Mop accumulates to high levels with increasing Mo
concentrations (Hoffmann et al. 2016).
<<<< <<<<<< >>>>>> >>>>

Mo-box consensus ATCGNTATATA–N6–TATATAACGAT
TSS
Rb. capsulatus anfA GTCGTTATATG–N7–TATATAACGGA

Rb. capsulatus mopA ATCGCTATTAG–N7–TATATAACGAT
TSS
Rp. palustris modE1 ACCGTTATATA–N7–CATATGACGAC
Rp. palustris anfA no Mo-box
Rp. palustris vnfA no Mo-box
Rs. rubrum anfA no Mo-box

Rs. rubrum modA no Mo-box
MopA MopA MopA
mopB mopA modABC anfA mop
Mo uptake Mo storage
MopB MopB
Fig. 5 Nitrogen fixation and molybdate transport-related Mo-boxes. Mo-boxes are highly con-
served palindromic sequences (marked by arrow heads) serving as binding sites for ModE-type
regulators (Studholme and Pau 2003). Conserved Mo-box nucleotides in the promoters of Rb.
capsulatus anfA and mopA, and Rp. palustris modE1 are highlighted in blue. Rb. capsulatus syn-
thesizes two ModE-like regulators, MopA and MopB, which repress transcription of the mopA-
modABC and anfA genes by binding Mo-boxes (red squares) overlapping the transcription start
sites (TSS) of mopA and anfA (Kutsche et al. 1996). In addition, MopA activates mop transcription
by binding the Mo-box (green square) preceding the mop TSS (Wiethaus et al. 2006)
While Mo represses mopA, the mopB gene is constitutively transcribed and

accordingly, the MopA/MopB ratio varies in response to Mo availability
(Hoffmann et al. 2016; Wiethaus et al. 2006, 2009). Under Mo-limiting condi-
tions, MopA is more abundant than MopB, whereas only MopB is left under
Mo-replete conditions. MopA and MopB form homodimers as well as het-
eromers (Wiethaus et al. 2009). Disruption of mopB enhances mop expression
suggesting that MopA-MopB heteromer formation counteracts mop activation
by MopA homodimers.
Since AnfA is essential for Fe-nitrogenase expression, anfA repression by MopA
and MopB prevents Fe-nitrogenase expression at high Mo concentrations (Fig. 3). In
contrast, Mo-nitrogenase levels increase with increasing Mo concentrations involving
a yet unknown post-transcriptional control mechanism (Hoffmann et al. 2014a, 2016).
In addition to ModABC, which imports molybdate at nanomolar concentrations
in the environment, Rb. capsulatus synthesizes the oxyanion transporter PerO, which
imports molybdate in micromolar ranges (Gisin et al. 2010). Besides molybdate,
16 B. Masepohl
PerO transports tungstate, vanadate, and sulfate. In contrast to the modABC genes,
transcription of perO is not repressed by molybdate.
Like Rb. capsulatus, Rp. palustris has two modE genes, one of which, modE1, clus-
ters with modABC genes, while the other is located at a distant position in the chromo-
some (Larimer et al. 2004). The modE1 promoter contains a likely Mo-box (Fig. 5)
indicating that ModE1 autoregulates its own expression in response to molybdate avail-
ability as is the case for Rb. capsulatus MopA. In contrast to the Rb. capsulatus anfA
promoter, the Rp. palustris anfA and vnfA promoters do not encompass an obvious
Mo-box suggesting that anfA and vnfA do not belong to the ModE1 regulon (see
below). Unlike Anabaena variabilis ATCC 29413, which synthesizes a high-affinity
vanadate transporter, VupABC, sustaining V-nitrogenase activity under vanadate-limit-
ing conditions, Rp. palustris lacks vupABC homologs (Pratte and Thiel 2006).
Disruption of the Mo-nitrogenase genes induces expression of V and
Fe-nitrogenases in Rp. palustris even at high molybdate concentrations otherwise
sufficient to repress Fe-nitrogenase in Rb. capsulatus (Oda et al. 2005; Wang et al.
1993). Similar to the situation in Rp. palustris, Rs. rubrum strains lacking active
Mo-nitrogenase express Fe-nitrogenase irrespective of Mo availability (Lehman and
Roberts 1991). Hence, the mechanisms controlling expression of the alternative
nitrogenases in Rp. palustris and Rs. rubrum differ from that in Rb. capsulatus.
Nitrogenase Protection Against Oxygen Damage
Mo, V, and Fe-nitrogenases are irreversibly damaged by oxygen (Blanchard and

Hales 1996; Chisnell et al. 1988; Gollan et al. 1993), and thus, many diazotrophs
synthesize nitrogenase only under anaerobic or microaerobic conditions. Other diaz-
otrophs have evolved different strategies to protect nitrogenase at high ambient oxy-
gen concentrations. Some filamentous cyanobacteria develop specialized N2-fixing
cells called heterocysts, which have thick cell walls limiting oxygen entry and lack
the oxygen-evolving photosystem PSII. Most rhizobia express nitrogenase exclu-
sively within special plant organs called nodules, in which oxygen partial pressure is
sufficiently low. Other strategies involve cytochrome bd oxidase (cydAB-encoded) or
the Shetna’s protein II (fesII-encoded) mediating “respiratory” and “conformational”
protection of nitrogenase, respectively, in A. vinelandii, Gluconacetobacter diazotro-
phicus, and Rb. capsulatus (Hoffmann et al., 2014a; Kelly et al. 1990; Moshiri et al.
1994; Schlesier et al. 2016; Ureta and Nordlund 2002). Conformational protection
depends on a ternary complex formed by FeSII, the Fe-protein, and the MoFe-protein
(Schlesier et al. 2016).
The Rb. capsulatus FeSII homolog, FdxD, supports diazotrophic growth via
Mo-nitrogenase (but not via Fe-nitrogenase) under semiaerobic conditions (Hoffmann
et al. 2014a). Expression of the fdxD gene, which is located immediately upstream of
the nifHDK genes, is activated by NifA1 and NifA2 but not by AnfA. Hence, the
fdxD gene belongs to the Mo-nitrogenase regulon, and its product specifically pro-
tects Mo-nitrogenase against oxygen damage.
NifA-dependent fdxD expression decreases with increasing oxygen concentra-

tions (Hoffmann et al. 2014a). This regulation is possibly explained by oxygen sen-
sitivity of NifA1 and NifA2, which belong to the class of oxygen-sensitive NifA
regulators (Fischer 1994; Paschen et al. 2001). Members of this class contain an
additional domain absent in oxygen-tolerant NifA proteins, the interdomain linker
domain, which is located between the central AAA and the C-terminal HTH domain.
The interdomain linker domain is implicated in metal (possibly Fe) binding and oxy-
gen or redox sensing in Bradyrhizobium japonicum and Herbaspirillum seropedicae
(Fischer et al. 1988, 1989; Oliveira et al. 2009).
Maximal fdxD expression requires both NifA1 and NifA2 (Hoffmann et al.
2014a), and maximal nifA2 expression depends on the two-component regulatory
system RegB-RegA (Elsen et al. 2000). In contradiction to the original assumption
that oxygen directly inhibits RegB kinase activity (Mosley et al. 1994; Sganga and
Bauer. 1992), the RegB-RegA system apparently responds to the cellular redox state
(Elsen et al. 2000). Besides controlling nitrogen fixation (via nifA2), the RegB-RegA
system regulates photosynthesis, carbon dioxide assimilation, and hydrogen oxida-
tion, thus acting as a master regulator of important energy-generating and energy-
consuming processes.
Nitrogenase Protection Against Carbon Monoxide Inhibition
Carbon monoxide (CO) inhibits all nitrogenase-catalyzed substrate reductions

except for proton reduction by blocking intramolecular electron flow and hence, CO
hampers N2 fixation and diazotrophic growth (Hwang et al. 1973; Lee et al. 2009;
Lockshin and Burris 1965; Rivera-Ortiz and Burris 1975; Shen et al. 1997; Yan
et al. 2012). A small protein, CowN, sustains N2-dependent growth of Rb. capsula-
tus and Rs. rubrum in the presence of CO (Hoffmann et al. 2014b; Kerby and
Roberts 2011). CowN has been suggested to form a complex with nitrogenase like
the Shetna protein but experimental evidence supporting this assumption is lacking
(Kerby and Roberts 2011). In both Rb. capsulatus and Rs. rubrum, cowN expression
is induced by CO, but cowN activation depends on different transcription activators
in these species.
CO induction of Rb. capsulatus cowN expression is mediated by the CO-responsive
regulator CooA (Hoffmann et al. 2014b), which belongs to the family of h eme-containing
transcription factors (Roberts et al. 2005). Expression of cooA is activated by NifA1 and
NifA2, whereas AnfA represses cooA and consequently, cowN. Accordingly, CowN
specifically sustains diazotrophic growth via Mo-nitrogenase but not Fe-nitrogenase-
dependent growth in the presence of CO.
The Rs. rubrum CooA homolog activates expression of CO dehydrogenase genes,
but is dispensable for cowN expression (Fox et al. 1996; Kerby and Roberts 2011;
Shelver et al. 1995). Instead, cowN expression in Rs. rubrum requires another
CO-responsive regulator, RcoM (Kerby et al. 2008; Kerby and Roberts 2011), which
is lacking in Rb. capsulatus.
18 B. Masepohl
Genes similar to cowN are widespread in bacteria (Kerby and Roberts 2011).
Apparently, all bacteria harboring a cowN homolog also possess nifHDK genes
implying that CowN-mediated Mo-nitrogenase protection is a common mechanism.
In contrast to strict ammonium repression of the nifHDK genes, however, cowN is
only partially repressed by ammonium in Rb. capsulatus and Rs. rubrum suggesting
that CowN function is not restricted to nitrogenase protection (Hoffmann et al.
2014b; Kerby and Roberts 2011).
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signals in Rhodospirillum rubrum. Microbiology 152:2075–2089
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Title: Holladayn juttu

Salapoliisiromaani
Author: Burton Egbert Stevenson
Release date: September 12, 2023 [eBook #71618]
Language: Finnish
Original publication: Hämeenlinna: Arvi A. Karisto Oy, 1943
Credits: Timo Ervasti and Tapio Riikonen
*** START OF THE PROJECT GUTENBERG EBOOK HOLLADAYN

JUTTU ***
HOLLADAYN JUTTU
Salapoliisiromaani
Kirj.
BURTON E. STEVENSON
Hämeenlinnassa, Arvi A. Karisto Oy, 1909.
SISÄLLYS:
I. Salama kirkkaalta taivaalta

II. Vaikuttavia asianhaaroja
III. Juttu kärjistyy
IV. Saan onnellisen päähänpiston
V. Syön päivällistä tuntemattoman kanssa
VI. Godfreyn ylistely
VII. Neiti Holladay tulee oikulliseksi
VIII. Salaperäinen kamarineiti
IX. Tutustun monsieur Martignyyn
X. Merkillinen katoaminen
XI. Riisun naamarin viholliseltani
XII. Cafe Jourdainissa
XIII. Matkalla
XIV. Osoittaudun huonoksi suojelusvartijaksi
XV. Kaksin on parempi kuin yksin
XVI. Leijonan pesässä
XVII. Etretat
XVIII. Huntu kohotetaan
XIX. Jutun loppu
I
Salama kirkkaalta taivaalta
Toimiston ilmakehä ei ollut tänä aamuna yhtä miellyttävä kuin

tavallisesti. Olimme kaikki saaneet raivata sinne tiemme läpi
myrskyn, lumen, ravan ja lumirännän, jollaista ei tapaa muualla kuin
keskellä maaliskuuta New Yorkissa, ja hyvä tuulemme oli tästä
kärsinyt. Minun oli ollut mahdotonta saada ajuria, ja raitiotiejunilla,
jotka yleensä olivat kovin myöhästyneet, vallitsi tietysti se tavallinen,
välttämätön tungos. Seurauksena tästä oli, että minä tulin konttoriin
puoli tuntia liian myöhään, ja astuttuani sisempään
konttorihuoneeseen sain hämmästyksekseni nähdä Grahamin,
vanhemman päällikkömme, jo istumassa työpöytänsä ääressä. Hän
nyökäytti jotenkin lyhyesti hyvää huomenta.
»Olisi hyvä, jos katsoisitte läpi nuo Hurdin juttua koskevat paperit,
Lester», sanoi hän ja ojensi ne minulle.
Otin ne ja istuuduin. Samassa temmattiin konttorin ulko-ovi taaskin

auki tavattoman rajusti.
En ollut koskaan nähnyt Roycea niin syvästi liikuttuneena, niin
ilmeisesti poissa suunniltaan kuin hän oli hyökätessään
silmänräpäystä myöhemmin luoksemme kädessään sanomalehti.
Hämmästyneenä sisääntulijan aikaansaamasta melusta käännähti
Graham pöytänsä ääressä ja tähysteli ihmeissään häntä.
»Mitä nyt, John?» alkoi hän. »Sinähän näytät kovin liikuttuneelta.

Mistä on kysymys?»
Royce levitti sanomalehden hänen eteensä pöydälle.
»Sinä et tietysti ole nähnyt aamulehtiä. Luepas tuo?» ja hän osoitti

vapisevalla sormella kirjoitusta, joka vei koko ensimmäisen sivun
ensimmäisen palstan — kunniasijan.
Huomasin, että Grahamin kasvojenilme muuttui heti hänen

luettuaan otsakkeen, ja kun hän oli ehtinyt kappaleen pitemmälle
varsinaiseen kertomukseen, näytti hän ihan kauhun valtaamalta.
»Tämä on ihmeellisintä, mitä olen koskaan lukenut!» puhkesi hän.
»Ihmeellistä!» huudahti toinen. »Se on enemmän kuin sitä, se on

halpamaista, alhaista! Ajatella, että niin hieno, sivistynyt tyttö kuin
Frances Holladay vakain tuumin murhaa oman isänsä — pistää
kylmäverisesti hänet kuoliaaksi — se on liian katalaa, liian
ihmeellistä, liian — liian…»
Hän keskeytti, melkein tukehtuen mielenliikutuksesta.
Hänen sanansa saivat minut äkkiä ojentautumaan tuolissani.

Frances
Holladay syytettynä — eipä ihme, ei! että Royce oli tuohtunut!
Mutta Graham jatkoi kirjoituksen lukemista toiseen kertaan,
tarkkaavammin, ja kun hän sitten nyökäytti päätään osoittaakseen,
että hän täysin käsitti kumppaninsa ajatuksen, muodostui hänen
kulmakarvojensa välille suora, syvä, hämmästystä ilmaiseva ryppy.
»Koko asia», sanoi hän vihdoin, »riippuu nähtävästi Rogersista —

Holladayn konttorin ensimmäisestä miehestä — ja sikäli kuin minä
tunnen Rogersia, on minun sanottava, että hän on maailmassa
viimeinen, joka esittäisi tahallansa vääriä väitteitä. Hän sanoo, että
neiti Holladay tuli isänsä konttoriin eilen iltapäivällä, viipyi siellä
kymmenen minuuttia ja poistui sitten kiireesti. Muutamia minuutteja
myöhemmin meni Rogers yksityiskonttoriin ja löysi päämiehensä
kuolleena. Siinä on koko asia, mutta se on kova pähkinä
purtavaksi.»
»Mutta siinä onnistutaan!» puuttui puheeseen toinen,

ponnistautuen saamaan takaisin ryhtinsä. »Minä otan tietysti jutun
huostaani.»
»Tietysti.»
»Neiti Holladay on arvattavasti lähettänyt sanan minulle eilisiltana,

mutta kuten tiedät, minä olin Babylonissa etsimässä sitä todistajaa
Hurdin juttuun. Miehestä on suuri hyöty, ja hänen todistuksellaan
tulemme voittamaan jutun. Mitä taas Brownin asiaan tulee, niin voi
vastaus odottaa huomiseen. Siinä kaikki, luullakseni.»
Graham nyökäytti myöntävästi.
»Niin — huomaan, että tutkinto alkaa kello kymmenen. Sinulla ei

ole pitkää aikaa viivytellä.»
»Ei ole. Ja haluaisinpa myöskin mielelläni saada jonkun mukaani
avuksi — jonkun, joka voisi olla minulle hyödyksi.» Ja hän heitti
silmäyksen minuun. »Voitteko te poistua täältä, Lester?»
Se kysymys tehtiin minulle. Tätä kysymystä juuri olinkin odottanut.
»Tietysti», vastasi Graham myöntyvästi. »Luonnollisesti —

sellaisessa asiassa kuin tämä. Annathan kai kuulla jotakin itsestäsi
ennen päivän loppua?»
Royce nyökäytti päätään ja meni ovelle.
»Sen teen. Ja me kyllä löydämme jonkun aukon Rogersin

kertomuksessa, uskokaa se! Tule, Lester!»
Otin mukaani kynän ja paperia ja seurasin häntä hissille. Hetkinen

sen jälkeen olimme kadulla. Nyt siellä oli yllin kyllin ajureita, jotka
jättivät kyydittäviänsä ja palasivat odotusasemilleen. Huusimme
luoksemme yhden, ja seuraavassa silmänräpäyksessä vierimme
määräpaikkaamme niin kovaa kyytiä kuin paha sää salli. Mielessäni
liikkui joukko kysymyksiä, joihin olisin mielelläni halunnut saada
vastauksen. Myrsky oli aamulla temmannut minulta sanomalehden
mukaansa, ja nyt minua harmitti, etten ollut toimeliaampi
hankkiakseni itselleni uutta. Silmäys seuralaiseeni osoitti
hyödyttömäksi koettaa saada mitään lähempiä selityksiä häneltä,
niin että tyydyin mietiskelemään sitä, minkä jo olin saanut tietää
Grahamilta.
Tunsin murhatun Hiram W. Holladayn oikein hyvin; ei ainoastaan

niinkuin jokainen newyorkilainen tunsi tämän monimiljonäärin
yhdeksi etevimmistä Wallstreetin liikemiehistä, vaan
mieskohtaisestikin yhtä hyvin, sillä hän oli yli kahdenkymmenen
vuoden ajan käyttänyt Graham & Roycea asianajajanaan. Hän oli
tähän aikaan lähemmäs seitsemänkymmenen vanha, vaikka hän
»kantoikin vuosiansa kunnialla»; hänen vaimonsa oli kuollut jo kauan
sitten, ja hänellä oli vain yksi ainoa lapsi, tytär Frances, joka oli kai
noin kaksikymmentäviisi vuotias. Tämä tytär oli syntynyt ulkomailla ja
oleksinut elämänsä ensi vuodet siellä yhdessä äitinsä kanssa, joka
oli jäänyt Rivieralle ja Italian ja Sveitsin vuoriseutuihin toivoen
saavansa, minun käsitykseni mukaan, jo tyttärensä syntymästä asti
heikon terveytensä takaisin. Viimein hän oli tullut kotiin yhdessä
mustasilmäisen tytön kanssa, ja ennenkuin vuosi oli kulunut
umpeen, hän oli kuollut.
Holladayn hellät tunteet olivat tästä hetkestä alkaen aivankuin

vahvistuneet ja keskittyneet tyttäreensä, joka oli kehittynyt pitkäksi,
kauniiksi tytöksi — aivan liian kauniiksi, kuten kohta ilmeni,
nuoremman päällikkömme mielentilalle. Hän oli saanut kohdata tytön
ensiksi liikeasioissa ja sitten ulkona seuraelämässä, ja kaikki me,
joilla oli silmät, voimme nähdä, miten hän vaipui yhä syvempään
kaihoon ja suruun, kun ei voinut toivoa koskaan voittavansa häntä;
sillä selväähän oli, että hänen isänsä piti häntä kyllin arvokkaana —
jota hän myöskin todella oli — solmimaan loistavan avioliiton. Väliin
luulin, että hän itsekin arvosti itsensä yhtä korkealle, sillä vaikka hän
oli alituisesti kosijalauman ympäröimänä, niin kukaan heistä ei, kuten
näytti, voinut saada häneltä pienintäkään mieltymyksen osoitusta.
Hän odotti, niin ajattelin — odotti; ja olin jo kuvitellut mielessäni sitä
julmaa pilaa, jota nuorempi päällikkömme saisi kärsiä, kun häntä
kerran vaadittaisiin laatimaan aviosopimus neiti Holladayn
naimisiinmenoa varten.
Vaunut pysähtyivät nytkähtäen, ja katsahdettuani ylös huomasin

tulleemme rikosoikeuden talolle. Royce hyppäsi rattailta, maksoi
kyydin ja juoksi ylös rappuja, minun seuratessani häntä. Hän
poikkesi käytävässä oikealle ja astui erääseen sen päässä olevaan
huoneeseen, jonka tiesin olevan tutkintotuomari Goldbergin
konttorin. Joukko ihmisiä oli jo kokoontunut sinne.
»Onko tutkintotuomari jo tullut?» kysyi seuralaiseni eräältä

konttoriapulaiselta.
»Kyllä. Hän on yksityiskonttorissaan.»
»Olkaa hyvä ja antakaa tämä kortti hänelle ja sanokaa, että

haluaisin tavata häntä heti, jos mahdollista.»
Kirjuri riensi kortteineen. Parin silmänräpäyksen kuluttua tuli hän

taas takaisin.
»Olkaa hyvä — tätä tietä», sanoi hän.
»Me seurasimme häntä läpi huoneen vastapäisellä seinällä

olevasta ovesta.»
»Ah, herra Royce, onpa hauska nähdä teitä», huudahti

tutkintotuomari astuttuamme sisään. »Koetimme eilisiltana saada
teidät käsiimme, mutta saimme vastauksen, että te olitte silloin
matkoilla, ja olin juuri soittamaisillani konttoriinne uudelleen…»
»Neiti Holladay on siis halunnut kutsua minut.»
»Niin, sen hän teki heti. Kun huomasimme, ettemme voineet

saada teistä tietoa, etsimme asioimistoverianne, mutta neiti Holladay
sanoi haluavansa odottaa siksi kunnes te palaatte.»
Voin nähdä, miten Royce punehtui tyydytyksestä.

»Toivoakseni ette ole katsonut tarpeelliseksi vangita häntä?» kysyi
hän.
»Emme ensinkään, häntä ei ole pienimmälläkään tavalla häiritty.

Hän on viettänyt yönsä kotona — valvonnan alla.»
»Se oli oikein. Onhan luonnollisesti mahdotonta epäilläkin häntä.»
Goldberg katsoi uteliaasti häneen.
»En tiedä, herra Royce», sanoi hän pitkäveteisesti. »Jos

todistukset tulevat olemaan sellaiset kuin minä luulen niiden tulevan,
niin on minun pakko pidättää hänet — yleinen syyttäjä vaatii sitä.»
Roycen kädet puristivat kovasti tuolin selustaa, ja ne vavahtivat

hänen kuullessaan tuomarin sanat.
»Yleinen syyttäjä tulee siis olemaan läsnä kuulustelussa?» kysyi

hän.
»Niin, odotamme häntä. Onhan tapaus mitä merkillisin, nähkääs.»
»Niinkö?»
»Niin se on meistä joka tapauksessa!» sanoi tutkintotuomari

kärsimättömästi.
Huomasin, että Roycella oli terävä vastaus kielellään, mutta hän

pidätti sen. Ei hyödyttänyt lainkaan loukata Goldbergiä.
»Haluaisin mielelläni tavata neiti Holladayta ennenkuin kuulustelu

alkaa», sanoi hän. »Onko hän täällä?»
»On, tässä viereisessä huoneessa. Saatte aivan heti mennä
hänen luoksensa. Julius, vie herra Royce neiti Holladayn luo!» lisäsi
hän kääntyen kirjuriin.
Voin vielä nytkin nähdä neiti Holladayn silmäini edessä, miten hän
kiihkoisasti nousi tuoliltaan, kun me astuimme sisään, ja käsitin heti,
että olin tuominnut häntä väärin. Hän otti pari askelta meitä kohti ja
ojensi rajusti molemmat kätensä; mutta jo seuraavassa
silmänräpäyksessä hän pidättäytyi ja laski ne ristiin eteensä.
»Voi, kuinka iloinen olen, kun tulitte!» puhkesi hän puhumaan niin
hiljaisella äänellä, että tuskin voin sitä kuulla. »Olen ikävöinyt teitä
kovin!»
»Suureksi ikäväkseni en ole voinut tulla ennemmin», sanoi

päällikköni vasten tahtoaan värähtävällä äänellä. »Minä — minä en
odottanut saavani nähdä teitä täällä ilman ainoatakaan…»
»Oh», keskeytti neiti, »ei ollut ketään, jota olisin halunnut mukaani.
Ystäväni ovat kyllä olleet hyvin kilttejä ja ystävällisiä — ovat
tarjoutuneet tekemään kaiken mahdollisen — mutta tunsin
tarvitsevani olla yksin ja ajatella. Kamarineitini olisin kyllä ottanut
luokseni, mutta…»
»Hän on todistajia, luullakseni», selitti Royce. »No niin, nyt kun

olen tullut tänne, niin aion viipyä täällä siksi, kunnes olen näyttänyt
toteen, kuinka perin naurettava tämä syytös teitä vastaan on.»
Hän istuutui jälleen tuolilleen ja katsoi Royceen vetoava ilme

mustissa silmissään.
»Luuletteko voivanne sen tehdä?» kysyi hän.

»Voivaniko? Luonnollisesti minä voin! Kysymyksessähän on niin
mahdoton väite, että sen täytyy kumoutua ilman muuta. Meidän
tarvitsee vain todistaa teidän alibinne — näyttää toteen, että te olitte
jossakin muualla silloin kun rikos tapahtui — ja silloin menee kaikki
sirpaleiksi. Senhän voitte tehdä helposti, vai kuinka?»
Väri oli taaskin kadonnut neiti Holladayn poskilta, ja hän kätki

kasvonsa käsiin.
»En tiedä», kuiskasi hän epäselvästi. »Tahdon ajatella. Voi, älkää

päästäkö asiaa niin pitkälle.»
Seisoin ihan hämmästyneenä. Hän halusi miettimisaikaa silloin,

kun hänen hyvä nimensä ja maineensa, ehkäpä elämänsäkin, olivat
pelissä! Voi nähdä, että päällikkönikin ihmetteli.
»Teen kaiken voitavani, että asia ei pääse niin pitkälle», sanoi hän
vitkaan. — »Minulla ei siis tule olemaan tilaisuutta kutsua teitä
todistajaksi omassa asiassanne — ja se vahingoittaa aina. Mutta
toivon, että asia yhtäkaikki päättyy heti — uskon sen. Missään
tapauksessa älkää olko huolissanne. Luotattehan minuun?»
Neiti Holladay katsoi taas — hymyillen — häneen.
»Luotan», sanoi hän hiljaa. »En voi toivoa itselleni parempaa

puolustajaa!»
Oli selvää, että jos Royce voittaisi tämän jutun, voittaisi hän
myöskin jotakin muuta. Luulenpa, että huoneen nurkassa oleva
poliisikonstaapelikin ymmärsi sen, sillä hän kääntyi pois hämillään,
mikä on harvinaista poliisille, ja meni katselemaan ulos ikkunasta.
En tiedä, millaisena päällikköni vastaus olisi tullut — hänen
huulensa värähtelivät, niin että hän toviin ei voinut puhua — sillä
samassa kuului koputus ovelta ja tutkintotuomarin kirjuri katseli
sisään.
»Olemme valmiit alkamaan», sanoi hän.
»Se on hyvä», vastasi Royce. »Tulen heti. Hyvästi niin kauaksi,

neiti
Holladay! Sanon teille vielä kerran, että voitte luottaa minuun.»
Sitten hän riensi ulos huoneesta niin voitonvarman näköisenä kuin

jos neiti Holladay olisi antanut hänelle voittamattoman aseen
mukaan taisteluun. Mutta sen sijaan, ajattelin itsekseni, hän olikin
sitonut Roycen kädet ja jalat, ennenkuin lähetti hänet areenalle.
II
Vaikuttavia asianhaaroja
Ulompi huone oli pakaten täynnä kansaa ja sen ilmakehä

tukehduttava ja vastenmielinen. Ainoastaan tutkintotuomarin pöydän
edessä olevan pienen aitauksen takana oli vähän väljää, ja me
istuuduimme siinä sijaitsevan pöydän ääressä oleville tuoleillemme
huoaten helpotuksesta.
Ei voi kuvitellakaan, kuinka monta sanomalehteä New Yorkissa on

läsnä jonkin tärkeän rikosasian käsittelyssä, joka kiinnittää niiden
lukijakunnan huomiota. Reportterit anastivat suurimman alan tuosta
pienestä huoneesta, paperia ja kyniä näkyi kaikkialla, ja eräässä
nurkassa seisoi mies mukanaan valokuvauskone, ottaakseen,
päättelyni mukaan, valokuvan neiti Holladaysta, jos hänet
kutsuttaisiin todistajapenkille, kun mitään hänen kuvaansa ei ollut
muulla tavoin saatavissa.
Näin Singletonin, yleisen syyttäjän, tulevan sisään ja asettuvan

tutkintatuomarin viereen, jonka jälkeen lautakunnan miehet
marssivat saliin huoneestansa ja asettuivat paikoilleen. Yritin tehdä
havaintoja heistä, yhdestä toisensa jälkeen, vähän levottomana,
mutta he näyttivät kaikki olevan valistuneita ja sangen hyvinvoipia
miehiä. Royce luki läpi heidän nimiluettelonsa ja pani huolellisesti
merkin jokaisen nimen kohdalle, sen mukaan kuin kirjuri luki heidät.
Sitten hän ojensi tutkintatuomarille listan pienellä nyökkäyksellä.
»Olkaa hyvä ja alkakaa!» sanoi hän. »He ovat luullakseni

täysilukuiset.
— Näyttäväthän ne kelvollisilta.»
»Se on hyvä lautakunta», vastasi tuomari, ottaen paperin.

»Parempi kuin tavallisesti. Oletteko valmis, herra Singleton?»
»Kyllä», sanoi yleinen syyttäjä. »Vaikka odottakaapas hetkinen»,

lisäsi hän, nousten ylös ja astuen meidän pöytämme luo. »Kutsutte
kai todistajaksi neiti Holladayn, otaksun…»
»Ja panen hänet alttiiksi kaikelle tälle?» Royce heitti silmäyksen

ympäri huonetta. »Ei, jos vain minun vallassani on estää se!»
»Minä en käsitä, miten te voisitte sen tehdä. Alibi on ainoa, mikä

voi pelastaa hänet tulemasta tuomituksi.»
»Aikaa on kyllä puhua siitä sitten kun tullaan niin pitkälle», vastasi
Royce. »Minä puolestani uskon, että kanne häntä vastaan raukeaa
kohta todistusten puutteessa.»
»Niin, jos se on mielipiteenne, niin…» Ja pureksien miettiväisenä

viiksiänsä meni Singleton takaisin pöytänsä ääreen.
Vaalinsa jälkeen, joka oli tapahtunut vuosi taaksepäin, hän oli

hankkinut erityisen maineen sekavien rikosjuttujen ratkaisijana ja oli
tuonut valoa kahteen, kolmeen huomiotaherättäneeseen
tapaukseen, jotka yhteen aikaan olivat uhanneet tehdä poliisin
älykkäisyydestä pilaa. Nyt hän vainusi todennäköisesti jotakin
samanlaista tässä jutussa, muutoin olisi hän siirtänyt sen jollekin
apulaiselleen. Lisättäköön kuitenkin, että vaikka hänen etevyytensä
olikin tehnyt hänet tavattoman suosituksi suuren yleisön
keskuudessa, niin hän oli kuitenkin etevyytensä ohella osoittanut
hänen asemassaan olevalle miehelle huonosti sopivaa käytöstä,
mikä saattoi virkaveljet katsomaan häntä vähän kieroon.
Hetkisen kuluttua nyökäytti hän tutkintotuomarille päätään, sisällä

olijoita kehoitettiin järjestykseen ja ensimmäinen todistaja kutsuttiin
sisälle.
Se oli Rogers, konttorin ensimmäinen mies. Tunsin tietysti hänet,

olin usein puhellut hänen kanssaan liikeasioista ja kunnioitin häntä
suuresti. Hän oli ollut Holladayn palveluksessa paljon kauemmin kuin
minä Graham & Roycella ja hänen maineensa oli, kuten Graham oli
viitannut, nuhteeton.
Tutkintotuomari Goldberg teki tavalliset valmistavat kyselynsä:

mikä nimi, ikä, missä asunto jne. Hän oli todellinen taituri
ristikuulustelun pidossa ja tuli pian asian ytimeen.
»Missä teidän työpöytänne sijaitsee Holladayn konttorissa?» kysyi

hän.
»Siellä on eteiskonttori, joka on konttoristeja varten, siitä tullaan

pienempään huoneeseen, jossa minun pöytäni on, ja minun
huoneestani vie ovi herra Holladayn yksityiskonttoriin.»
»Onko Holladayn huoneessa mitään muuta ovea?»
»Ei.»
»Oliko mahdollista päästä sinne ikkunain kautta?»
»Ei, ne ovat kadun puolella, ja konttorimme on kahdeksannessa

kerroksessa.»
»Ja paloportaat?»
»Ne ovat rakennuksen perällä — kadun puolelta ei mene mitään

paloportaita — siellä on vain kohtisuora seinä.»
»Niin että sen, joka tulee sisään tai menee ulos yksityiskonttorista,
on ehdottomasti kuljettava teidän työpöytänne ohitse?»
»Niin, aivan ehdottomasti.»
»Olisiko kukaan voinut kulkea teidän ohitsenne näkemättänne?»
»Ei, se olisi ollut ihan mahdotonta.»
Tutkintotuomari oikaisi selkänsä tuolin selustaa vastaan. Yhdestä

asiasta oltiin siis päästy selvyyteen.
»Herra Rogers», sanoi hän, »olkaa nyt hyvä ja puhukaa meille

omalla tavallanne ja niin seikkaperäisesti kuin mahdollista, mitä
tapahtui konttorissanne kello vähää vaille viisi eilisiltana!»
Näin, että Rogers oli hermostuksissaan. Hänen kasvonsa olivat

kalpeat, hän kostutti lakkaamatta hermostuneesti huuliansa ja puristi
vavahdellen käsillään tuolinsa käsinojaa. Hänen kertomuksensa oli
kaikkea muuta kuin miellyttävä.
»Niin», alkoi hän, »meillä oli kova kiire eilen, ja viivyimme

konttorissa melkoista myöhempään kuin tavallisesti, mutta viiden
aikaan olimme lopettaneet päivän työn, ja kaikki konttoristit menneet

Textbook Modern Topics in The Phototrophic Prokaryotes Metabolism Bioenergetics and Omics 1St Edition Patrick C Hallenbeck Eds Ebook All Chapter PDF

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Textbook Modern Topics in The Phototrophic Prokaryotes Metabolism Bioenergetics and Omics 1St Edition Patrick C Hallenbeck Eds Ebook All Chapter PDF

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Modern Topics in the Phototrophic

Prokaryotes Metabolism Bioenergetics

Topics in Modern Differential Geometry 1st Edition

Sequence Spaces-Topics in Modern Summability Theory 1st

MKSAP Neurology Patrick C. Alguire (Editor In Chief)

Big Data in Omics and Imaging Association Analysis 1st

Modern C 1st Edition Jens Gustedt

Modern C 1st Edition Jens Gustedt

Hydrogen Peroxide Metabolism in Health and Disease 1st

From Random Walks to Random Matrices - Selected Topics

Modern Topics in the

ISBN 978-3-319-51363-8 ISBN 978-3-319-51365-2 (eBook)

Library of Congress Control Number: 2017933463

© Springer International Publishing AG 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

 egulation of Nitrogen Fixation in Photosynthetic Purple

Abstract Biological nitrogen fixation (BNF) is the nitrogenase-catalyzed process in

© Springer International Publishing AG 2017 1

represses anfA transcription and hence, Fe-nitrogenase expression in Rb. capsulatus,

Keywords Nitrogen fixation • Nitrogenase • Rhodobacter • Rhodopseudomonas •

Introduction to Biological Nitrogen Fixation

N 2 + 8 e − + 8 H + + 16 ATP → 2 NH3 + H 2 + 16 ( ADP + Pi ) (1)

All N2-fixing (diazotrophic) bacteria and archaea possess a molybdenum-­

 rganization of Nitrogen Fixation Genes in Purple Nonsulfur

Regulation Nitrogenase Cofactor synthesis Electron transport Unknown function

 ascade Activation of Nitrogen Fixation in Rhodobacter

Growth with NH4+ NtrC inact ive

N2-f ixing conditions NtrC P

D nifA1 D nifA2 D mopA modABC D anfA

NifA1 NifA2 MopA AnfA

case in other proteobacterial diazotrophs (Hübner et al. 1991; Schüddekopf et al.

 mmonium Inhibition of Nitrogen Fixation in Rhodobacter

Pierrard et al. 1993a; Yakunin and Hallenbeck 1998b). Ammonium-induced nitroge-

 mmonium Regulation of Nitrogen Fixation

Expression and activity of Mo-nitrogenase in Rp. palustris is regulated at three lev-

N2-f ixing conditions NH4+switch-of f NH4+consumed

AmtB AmtB AmtB

GlnK UMP GlnK GlnK UMP

GlnB UMP GlnB GlnB UMP

DraG DraT inactive

Fe-protein DraT Fe-protein DraG Fe-protein

MoFe-protein MoFe-protein MoFe-protein

Fig. 4 Model of ammonium-responsive nitrogenase regulation in Rb. capsulatus. Upon ammo-

activity requires GlnK2, whose expression depends on NtrC, which is synthesized

 mmonium Regulation of Nitrogen Fixation in Rhodospirillum

vated by membrane sequestration involving AmtB1, unmodified GlnJ, and possibly

Darkness Regulation of Nitrogenase

Like ammonium addition, light deprivation causes nitrogenase switch-off in photo-

Iron Regulation of Electron Transport to Nitrogenase

Regulation of Molybdate Uptake and Alternative Nitrogenases

Most bacteria synthesize high-affinity molybdate transporters (modABC-encoded)

<<<< <<<<<< >>>>>> >>>>

Rb. capsulatus anfA GTCGTTATATG–N7–TATATAACGGA

Rs. rubrum anfA no Mo-box

MopA MopA MopA

mopB mopA modABC anfA mop

While Mo represses mopA, the mopB gene is constitutively transcribed and

Nitrogenase Protection Against Oxygen Damage

egulation of Nitrogen Fixation in Photosynthetic Purple

Introduction to Biological Nitrogen Fixation

All N2-fixing (diazotrophic) bacteria and archaea possess a molybdenum-

rganization of Nitrogen Fixation Genes in Purple Nonsulfur

ascade Activation of Nitrogen Fixation in Rhodobacter

mmonium Inhibition of Nitrogen Fixation in Rhodobacter

mmonium Regulation of Nitrogen Fixation

mmonium Regulation of Nitrogen Fixation in Rhodospirillum

Darkness Regulation of Nitrogenase

Iron Regulation of Electron Transport to Nitrogenase

Regulation of Molybdate Uptake and Alternative Nitrogenases

Nitrogenase Protection Against Oxygen Damage

Nitrogenase Protection Against Carbon Monoxide Inhibition