Download tethering redox proteins to the outer membrane in Neisseria and

ICoN2 and the NCycle16
Tied down: tethering redox proteins to the outer
membrane in Neisseria and other genera
Xi Li, Steven Parker, Manu Deeudom1 and James W. Moir2
Department of Biology, University of York, Heslington, York YO10 5DD, U.K.
Abstract
Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic
membrane or in the periplasm. An alternative arrangement appears to be widespread within the
betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with
the outer membrane. In the present paper, we discuss the structural properties of these outer membrane
redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox
proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and
the redox protein globular domain that should enable the redox cofactor-containing domain to extend
from the outer membrane, across the periplasm and towards the inner membrane. It is argued that
the constraints imposed on the movement and orientation of the globular domains by these tethers
favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer
membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the
host–microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer
membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this
organizational arrangement may be more common than previously recognized.
Introduction
Parts of bacterial respiratory chains are, by necessity,
embedded in the cytoplasmic membrane in order to allow
generation of ion gradients to drive the synthesis of ATP.
In Gram-negative bacteria, it is typical to also find redoxcofactor-containing respiratory proteins within the periplasm
where they may function as electron carriers or redox
enzymes. Many of the enzymes and electron carriers of,
for example, denitrification are typically localized within the
periplasm. A variant on this organizational structure is found
in the genus Neisseria, in which a number of redox proteins
are predicted to be associated with the outer membrane.
The width of the periplasm (∼170 Å [1,2], where 1 Å =
0.1 nm) presents a potential problem for the movement of
electrons from the inner membrane respiratory complexes to
the outer membrane electron carriers/respiratory electronacceptor reductases. In the present paper, we review (i) the
evidence for association of respiratory proteins in Neisseria
with the outer membrane, (ii) the structural properties of the
outer membrane respiratory proteins and the consequences
of this for their functionality, (iii) additional possible roles
for outer membrane attachment in the lifestyle of Neisseria
species in their natural habitat, and (iv) the evidence that outer
membrane-tethered redox proteins are also found outside the
genus Neisseria.
Key words: cupredoxin, cytochrome, electron transfer, Neisseria, periplasm, redox protein
tethering.
Abbreviations used: AniA, anaerobically induced protein A; CCP, cytochrome c peroxidase; GST,
glutathione transferase; Laz, lipid-modified azurin; LCR, low-complexity region.
1
Present address: Department of Microbiology, Faculty of Medicine, Chiang Mai University,
Thailand.
To whom correspondence should be addressed (email [email protected]).
2
Biochem. Soc. Trans. (2011) 39, 1895–1899; doi:10.1042/BST20110736
The outer membrane redox proteins of
Neisseria
The genus Neisseria (which belongs to the Betapreoteobacteria) consists of two pathogenic species (Neisseria
meningitidis and Neisseria gonorrhoeae) and about 20
commensal species, which typically inhabit epithelial mucosal
surfaces. Many are colonists of the human epithelial mucosa
in the mouth and upper respiratory tract. Under oxygenlimited conditions, Neisseria species can denitrify nitrite [3,4],
a process that relies on the expression of AniA (anaerobically
induced protein A) [5–7], formerly known as Pan1 [8].
AniA is a copper-type nitrite reductase (NirK) [6,9], but is
distinguished by being an outer membrane protein (based
originally on selective solubilization of the cytoplasmic
membrane using dodecyl sarcosinate [10]), rather than a
periplasmic protein as is typical for NirK in other Gramnegative organisms. AniA was originally identified from N.
gonorrhoeae [10], but is also present in outer membranes
from other Neisseria species [11]. The association with
the outer membrane is via a palmitate residue, which is
covalently attached to the N-terminal cysteine residue that
immediately follows the cleaved signal peptide [7]. Since
localization of AniA was only determined biochemically
using differential detergent-solubilization methodology, we
set out to investigate localization of AniA in the commensal
Neisseria lactamica, by isolation of intact membranes and
separation by sucrose gradient centrifugation (Figure 1). The
experiments provide useful confirmation that AniA [and Laz
(lipid-modified azurin), see below] are found in the outer
membrane, whereas the c-type cytochromes are associated
with the inner membrane.
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Figure 1 Localization of redox proteins in membranes in
Neisseria
N. lactamica cells were fractionated into periplasm, cytoplasm and
membranes using lysozyme treatment and osmotic shock. Localization
of proteins into the inner membrane (IM) or outer membrane (OM)
fractions are shown in panels (A–D). (A) The major band from a
Coomassie Blue-stained gel is a porin, only seen in outer membranes.
(B) Western blot with anti-AniA antibodies shows that AniA is mainly
associated with outer membrane. (C) Western blot with anti-Laz
Figure 2 N. meningitidis Laz is enriched in outer membrane blebs
N. meningitidis was fractionated into periplasm, cytoplasm and
membranes using lysozyme treatment and osmotic shock, and outer
membrane-containing blebs were prepared. Samples containing total
cell extract (lane 1), blebs (lane 2), total membranes (lane 3), periplasm
(lane 4) and cytoplasm (lane 5) are shown. Upper panel: Ponceau S
staining; lower panel: probed with anti-Laz antibody. Molecular masses
are indicated in kDa.
antibodies shows that Laz is mainly associated with outer membrane.
(D) Haem staining shows that c-type cytochromes are mainly associated
with the inner membrane (48 kDa, CcoP; 30 kDa, cytochrome c5 /PetC;
24 kDa, CcoO/cytochrome c4 ; 16 kDa, cytochrome c’).
In addition to a lipid-associated copper-type nitrite
reductase, a Laz-like protein is found in Neisseria species [12–
14]. Azurins are copper-containing electron-carrier proteins
found in many bacteria. The role of Neisseria azurin as
an electron-transfer protein is unclear, but it may have an
important role in the host–microbe interaction (see below).
Like AniA, Laz consists of a signal sequence followed
immediately by a cysteine residue which becomes covalently
associated with outer membrane lipid. Using antibodies
raised against Laz protein, we found that Laz is indeed
associated with the outer membrane (Figure 1) or with outer
membrane vesicles or blebs (Figure 2).
Two other outer membrane-associated redox proteins are
the CCP (cytochrome c peroxidase), which is found in N.
gonorrhoeae, and not in most other Neisseria species [15],
and the cytochrome c [16]. Again, homologous proteins
from other genera are typically found as soluble proteins in
the periplasm, but in Neisseria, they are associated with
membrane.
Flexible tethers for attachment to the
outer membrane
The Neisseria outer membrane respiratory proteins AniA,
Laz and CCP share common organizational features. The N
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is recognized by the globomycin-sensitive signal peptidase II
[15]. This yields a mature peptide with an N-terminal cysteine
followed by an extended LCR (low-complexity region),
which is in turn followed by a protein domain with high
identity with the globular domains of homologous nitrite
reductase, azurin and CCP respectively. {We have excluded
cytochrome c from this group, since (i) it appears to lack
an LCR, and (ii) its location in the cell is questionable;
although the Neisseria cytochrome c’ is an outer membrane
protein when heterologously expressed in Escherichia coli
[16], it appears to be associated with the inner membrane
in N. lactamica and N. meningitidis (W.M. Huston, X. Li
and J.W. Moir, unpublished work).} The LCR seems to
be a Neisseria genus-specific addition to these proteins and
consists of a stretch of 30–40 amino acids, rich in alanine
(∼50% of the amino acid composition of LCR is alanine),
proline, glutamate, glutamine, serine and threonine (Figure 3).
The LCR in Laz is particularly similar to a repeated sequence
motif (AAEAP) found in the outer membrane Lip protein,
where the sequence is known as the H.8 antigen [17]. We
predict that the LCR forms an extended unstructured region
that acts as a flexible linker, attaching the globular domains
of the proteins to the outer membrane, but enabling the
proteins to be located at some distance from the membrane.
It is not possible to be sure of the extension lengths of
these linker/LCR domains in the absence of experimental
support, but it would appear likely that the translational
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Figure 3 Putative flexible linker regions/LCRs from Neisseria
outer membrane-associated redox proteins
The regions shown begin after the predicted signal peptidase II cleavage
site (which leaves an N-terminal cysteine residue) and end at the
beginning of the predicted globular domain of the redox protein in
question (deduced by comparison with homologous, but periplasmic,
members of the same family). Amino acids alanine, glutamate, proline,
serine and threonine are shown in bold to emphasize that most of the
amino acids from the linker region belong to these types.
donor to AniA [20,21]. We have argued previously that
the tether regions of AniA should allow it to extend
sufficiently from the inner leaflet of the outer membrane to
form a complex with the inner membrane di-haem protein
cytochrome c5 [21], so long as the tethers can extend up to
∼60 Å, which is within the predicted range (50–90 Å, see
above) (Figure 4).
Why have Neisseria adapted to attach
respiratory proteins to the outer
membrane via flexible tethers?
To limit protein dynamics and enhance
formation of electron-transfer complexes
distance between adjacent amino acids along the peptide chain
will be in the region between 1.5 and 3 Å (the translational
distances between amino acids in the α-helix and in collagen
respectively). From this, we estimate that the tethers can allow
the globular domains of AniA, Laz and CCP to be ∼50–90 Å
from the surface of the inner leaflet of the outer membrane.
Hong et al. [18] showed that the Laz protein or a GST
(glutathione transferase) fused to the signal peptide plus Nterminal LCR of Laz enables the globular azurin–GST to
be surface-exposed in Neisseria (or indeed on expression in
E. coli). Presumably, in this case, (at least some of) Laz is
covalently linked to lipid in the outer leaflet of the outer
membrane. The alternative explanation would be for the LCR
to cross the outer membrane; this seems unfeasible, given that
it is a highly charged peptide chain. Surface exposure of Laz
(or other redox proteins) may be important for cell biological
reasons, but it is hard to reconcile an external location for
these outer membrane redox proteins with their function
in respiration. There are no candidate electron-transporting
outer membrane proteins in Neisseria that might enable
electrons to pass from the periplasm out to an extracellularly
located respiratory reductase, such as AniA or CCP. Thus we
reason that a subpopulation of these outer membrane proteins
may flip across to the outer leaflet of the outer membrane, but
that the population that function in respiration are tethered
to the inner leaflet of the outer membrane.
A further reason for presuming that the AniA (and CCP)
enzymes are located within the periplasm (although tethered
to the outer membrane) lies in the overall similarity of
their globular domains to their homologues from other
organisms. There are no gross differences in the structure
of AniA compared with other NirK homologues, indicating
that it receives electrons via formation of a direct electrontransfer complex directly with a cytochrome or cupredoxin,
as seen for its relatives from, e.g., Achromobacter xylosoxidans
[19]. Any putative outer membrane pore that enabled this
interaction would need to have an internal pore diameter
of ∼30 Å to house a cytochrome/cupredoxin domain.
In N. meningitidis, mutation of the gene encoding an
inner membrane cytochrome c5 ablates nitrite reduction,
supporting a role for this cytochrome as the sole electron
In a system where redox proteins are free to move in
the periplasm, there is effectively free tumbling in the
aqueous milieu, although diffusion rates may be slow due
to constrained space, since the compartment has a high
concentration of biomolecules including the cross-linked cell
wall polymer peptidoglycan. Tethering of a protein to the
outer membrane will limit its capacity for lateral movement,
as it would need to move both the membrane anchor and
the globular protein domain in a way that would avoid
becoming tangled in the peptidoglycan. Furthermore, and
more importantly for electron transfer, tethering may reduce
the capacity for tumbling. The AniA protein is a trimer
[9], and thus in N. meningitidis, it is tethered to the outer
membrane by three distinct peptides. The enzyme structure
approximates to that of a triangular prism with a height of
∼50 Å and triangular sides of ∼70 Å. The three tethering
peptides will thus prevent the enzyme from being able to
rotate freely (because the tethers are not long enough to
wrap around the enzyme). The tethers are attached to the
face of the protein opposite the electron-accepting face,
thus the tethers ensure that the AniA enzyme is maintained
in a position in which the redox-accepting sites face the
cytoplasmic membrane. The tethers may also thread through
the peptidoglycan within the periplasm, further reducing
mobility of the tethered enzyme. The cytochrome c5 electron
donor is associated with the cytoplasmic membrane via
a predicted membrane-spanning α-helix. The constrained
nature of both donor and acceptor favour the formation
of an electron-transfer complex. Essentially, the tethering
lowers the loss of entropy that would be associated with
the formation of a complex between a membrane-bound
protein and a freely tumbling protein in aqueous solution,
since, in the current example, both proteins already have
a significantly deceased number of degrees of freedom
compared with freely tumbling proteins. Thus there is a
thermodynamic explanation for the tethering. The decreased
dynamics of the proteins and the tendency to be appropriately
oriented for complex formation should also translate into a
kinetic advantage as the likelihood of an appropriate collision
between the two partners is increased.
The existence of tethered enzymes seems to be uncommon
(but see below), and so there presumably is some
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Figure 4 Arrangement of AniA and cytochrome c5 between the inner and outer membranes in Neisseria
The AniA structure is based on the structure of AniA from N. gonorrhoeae [9]. The cytochrome c5 structure was modelled
based on the structure of the di-haem cytochrome c4 from Pseudomonas stutzeri [27].
disadvantage to this organizational strategy. The limited
dynamic movement of the AniA protein favours its
interaction with cytochrome c5 as electron donor, but
the disadvantage may be a lack of flexibility to form
interactions with different electron-carrying partners in a
flexible branched respiratory chain.
Tethering of redox proteins for reasons related
to Neisseria lifestyle
An alternative to the biochemical arguments for redox protein
tethering may relate to the lifestyle of Neisseria species and
moonlighting functions for proteins such as Laz and AniA.
Soluble periplasmic homologues of Laz, such as the azurin
from Pseudomonas aeruginosa have been shown to have roles
in apoptosis, in particular of cancer cells (see, e.g., [22]).
N. meningitidis Laz has also been shown to be competent
at entering glioblastoma cells and causing a high level of
cytotoxicity [18]. This ability of Laz to enter mammalian
cells is related to its lipid attachment and exposure to the
surface of the cell [18]. Recently, it has been shown that
antibodies that specifically recognize Laz (or another outer
membrane protein Lip) are able to block serum-dependent
killing of meningococcal cells, and that these antibodies
recognize sequences in the LCR [23]. Evidence has also been
presented that AniA (from N. gonorrhoeae) confers serum
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reductase activity, indicating that this protein might also
have a moonlighting role in subversion of the host immune
response against Neisseria [24].
Tethered redox proteins outside Neisseria
A standard organizational strategy in Gram-positive bacteria
is the tethering of extracellular proteins such as homologues
of redox proteins that are found in the periplasm in Gramnegative bacteria, and will not be discussed in the present
paper. What is notable, however, is that bioinformatic
searches reveal that tethering of redox proteins may also be
fairly widespread among Gram-negative bacteria belonging
to proteobacterial phylum.
Nitrite reductases have been demonstrated to be commonly distributed by horizontal gene transfer [25,26].
Closely related to the Neisseria AniA nitrite reductases are
the nitrite reductases from Gram-positive members of the
Flavobacteria class, which is part of the Bacteroidetes phylum.
These flavobacterial nitrite reductases are predicted to be
membrane-associated lipoproteins, as would be expected
for organisms with a single membrane. More interesting is
that predicted lipid-tethered copper-type nitrite reductases
are also found within the gammaproteobacterial genera
Moraxella and Psychrobacter. Although there is no detectable
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conservation with the Neisseria LCR region, there is a
region of 35 amino acids between the predicted N-terminal
cysteine residue of the mature protein and the beginning
of a recognizable nitrite reductase globular domain. The
copper-type nitrite reductases from the gammaproteobacteria
Moraxella and Psychrobacter are most closely related to those
from the betaproteobacterium Neisseria, indicating that the
outer membrane tethering may have arisen just once for this
enzyme within the proteobacteria.
Lipid-modified CCPs are also observed outside Neisseria
species. CCP homologues within the deltaproteobacterial
genera of Myxococcus, Stigmatella and Haliangium all contain
a CCP with predicted N-terminal lipoprotein-attachment
sites. Again the predicted N-terminal cysteine residue is
followed by around 35 amino acids before the beginning
of the CCP globular protein structure. The CCP enzymes
from the Deltaproteobacteria are not particularly closely
related to Neisseria CCP; closer relatives being found in such
phyla as Aquificae and the cyanobacteria, suggesting that this
strategy for localizing CCP may have arisen more than once.
We have not yet been able to identify lipid-modified
azurins outside the Neisseriaceae, but they are found in the
genera Eikenella, Simonsiella and Kingella, which are closely
related to the genus Neisseria.
It would appear that diverse organisms employ tethering
of redox proteins to their (outer) membranes in organisms
with the classical two-membrane Gram-negative-style cell
envelope. It will be of interest to determine precisely
how widespread this phenomenon is and look for further
experimental evidence on this mode of organizing respiratory
chains.
Acknowledgements
We thank Dr Jennifer Potts for helpful discussions.
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Received 2 September 2011
doi:10.1042/BST20110736
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