Int. J. Mol. Sci. 2012, 13, 8038-8050; doi:10.3390/ijms13078038
International Journal of
From Evolution to Pathogenesis: The Link Between β-Barrel
Assembly Machineries in the Outer Membrane of Mitochondria
and Gram-Negative Bacteria
Jhih-Hang Jiang, Janette Tong, Kher Shing Tan and Kipros Gabriel *
Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus,
Melbourne 3800, Australia; E-Mails: firstname.lastname@example.org (J.-H.J.);
email@example.com (J.T.); firstname.lastname@example.org (K.S.T.)
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +61-3-9902-9213; Fax: +61-3-9905-3726.
Received: 1 May 2012; in revised form: 21 June 2012 / Accepted: 21 June 2012 /
Published: 28 June 2012
Abstract: β-barrel proteins are the highly abundant in the outer membranes of
Gram-negative bacteria and the mitochondria in eukaryotes. The assembly of β-barrels is
mediated by two evolutionary conserved machineries; the β-barrel Assembly Machinery
(BAM) in Gram-negative bacteria; and the Sorting and Assembly Machinery (SAM) in
mitochondria. Although the BAM and SAM have functionally conserved roles in the
membrane integration and folding of β-barrel proteins, apart from the central BamA
and Sam50 proteins, the remaining components of each of the complexes have diverged
remarkably. For example all of the accessory components of the BAM complex characterized
to date are located in the bacterial periplasm, on the same side as the N-terminal domain of
BamA. This is the same side of the membrane as the substrates that are delivered to the
BAM. On the other hand, all of the accessory components of the SAM complex are located
on the cytosolic side of the membrane, the opposite side of the membrane to the
N-terminus of Sam50 and the substrate receiving side of the membrane. Despite the
accessory subunits being located on opposite sides of the membrane in each system, it is
clear that each system is functionally equivalent with bacterial proteins having the ability
to use the eukaryotic SAM and vice versa. In this review, we summarize the similarities
and differences between the BAM and SAM complexes, highlighting the possible selecting
pressures on bacteria and eukaryotes during evolution. It is also now emerging that
Int. J. Mol. Sci. 2012, 13 8039
bacterial pathogens utilize the SAM to target toxins and effector proteins to host
mitochondria and this will also be discussed from an evolutionary perspective.
Keywords: β-barrel proteins; mitochondria; bacteria; outer membrane; protein transport;
protein folding; evolution
1. Basic Features of the Bacterial and Mitochondrial Membranes
Gram-negative bacteria contain two lipid bilayers which envelope the periplasm: the inner
membrane (IM) and outer membrane (OM). The asymmetric outer membrane is composed of
lipopolysaccharides (LPS) in the outer leaflet and phospholipid in the inner leaflet while the symmetric
inner membrane is composed of a phospholipid bilayer. The outer membrane predominantly harbors
β-barrel fold proteins whereas α-helical based transmembrane proteins are primarily found in the inner
membrane. The outer membrane provides the physical barrier from the outside world, but also must
maintain selective permeability for the uptake of nutrients to support physiological functions, for the
release of virulence factors and in some cases for multidrug resistance. This is achieved by the outer
membrane proteins [1,2]. Evolutionarily, mitochondria are derived from an ancient α-proteobacterium
through an endosymbiotic relationship that was initiated billions of years ago [3–5]. They have
retained their double membrane structure that forms the inner and outer membranes, which enclose
two aqueous environments, the intermembrane space and the matrix. This allows for the separation
of biochemical processes and hence mitochondria perform many roles in eukaryotic cells. To carry
out these functions, nutrients, biochemical intermediates, lipids and proteins must traverse the
mitochondrial membranes. Outer membrane proteins (OMPs), particularly β-barrel proteins, play a key
role in these transport processes .
2. β-Barrel Proteins and the Assembly in the Bacterial and Mitochondrial Outer Membrane
β-barrel proteins are defined as anti-parallel β-strands forming larger β-sheets that are connected
through hydrogen bonds between the transmembrane strands that confer extreme stability . As most
β-barrels are membrane spanning proteins, it is typical for them to possess amino acids with
hydrophobic side chains on their surface that abuts the phospholipids, while hydrophilic or charged
residues often face the central cavity [1,7,8].
The assembly of β-barrel OMPs is mediated by sophisticated molecular machineries.
In Gram-negative bacteria, nascent polypeptides are synthesized with an amino-terminal signal peptide
by cytoplasmic ribosomes. These precursors are bound by factors such as SecB and SecA or the
chaperone DnaK and transported to the secretion machinery (Sec). The Sec translocates proteins into
the periplasm. Once in the periplasm, the signal peptide is processed, and precursor proteins associate
with the chaperones SurA and Skp which not only transfer proteins to the BAM complex for outer
membrane insertion but also prevent their aggregation . A recent study has also shown that
chaperones also play an important role in assisting folding and assembly of β-barrel precursor proteins,
using in vitro reconstitution techniques with BAM and its substrates .
Int. J. Mol. Sci. 2012, 13 8040
Similarly, in mitochondria, precursor β-barrel proteins are also synthesized by cytosolic ribosomes
and are bound by chaperones in the cytosol . These precursor substrates are transported to the
receptor subunits of the Translocase of the Outer Mitochondrial membrane (TOM). The TOM complex
translocates substrate proteins across the outer membrane into the intermembrane space. The small
chaperones Tim9/10 or Tim8/13 associate with substrates, in a manner which is functionally analogous
to that performed by SurA and Skp in bacteria, for the transfer to the Sorting and Assembly Machinery
(SAM) complex at the mitochondrial outer membrane [12–17].
3. The β-Barrel Assembly Machinery in Gram-Negative Bacteria
In bacteria, the Omp85 family of protein assembly machines is the key and essential component of
outer membrane β-barrel protein insertion processes. Omp85 protein homologues are found in bacteria
and eukaryotes, including fungi, plants (mitochondria and chloroplasts) and animals [15–19]. Study of
the BamA protein in N. meningitidis  and Escherichia coli [20–22] first revealed the essential role
of Omp85 protein machines in folding and assembly of outer membrane proteins. BamA is highly
conserved throughout all Gram-negative bacterial species . Structural predictions relating to BamA
suggest that it is composed of two parts: a large soluble N-terminal domain protruding to the periplasm;
and the carboxy-terminal β-barrel domain sitting in the OM. The large soluble domain has five repeat
POTRA (polypeptide transport-associated) domain, these repeat domains are suggested to bind
unfolded β-barrel proteins . The deletion of each POTRA domain exerts different effects to
complex stability and substrate binding. POTRA domains 2 to 4 are important for the interaction with
the partner protein in the BAM complex, BamB, whereas deletion of POTRA 5 specifically affects
association with other lipoprotein partners [24,25].
Defects in BAM complex function have many deleterious primary and secondary effects, including
accumulation of unfolded protein substrates and reduced LPS and phospholipid incorporation into the
outer membrane. Failures in these processes would cause accumulation of these factors at the inner
membrane . Recent evidence indicates BamA acts as the assembly hub for the formation of an
LptD/E complex in the outer membrane, which is subsequently essential for LPS biogenesis .
In E. coli, BamA (previously referred as YaeT) is associated with four lipoproteins BamB, C, D and E
(previously known as YfgL, NlpB, YfiO and SmpA respectively) [20,28–30]. The BAM is also
involved in autotransporter protein biogenesis, which is not surprising as autotransporters have a
C-terminal β-barrel domain [31,32]. The N-terminus of autotransporter proteins consist of passenger
domains which are translocated through the folded barrel formed by the C-terminus and usually act as
virulence factors during infection. The BAM was shown to be associated with stalled autotransporter
substrates created by an in vivo site-specific photocrosslinking approach. It is hypothesized that the
β-barrel domain initially interacts with chaperones in the periplasm and then passed to BamA, BamB
and BamD sequentially. As both BamB and BamD are still found crosslinked to substrates at later time
points during chase experiments, it has been suggested that they are involved in the late stages of
folding/assembly. The passenger domain of autotransporter EspP can transiently interact with the
channel BamA and the release of passenger domain seems to be a checkpoint for the completion of
assembly of β-barrel domain [31,32].
Int. J. Mol. Sci. 2012, 13 8041
4. The Sorting and Assembly Machinery (SAM) in Mitochondria
The key molecule of the eukaryotic SAM is the β-barrel core protein, Sam50, the homolog of the
bacterial protein BamA. Sam50 is predicted to have one POTRA domain that faces the mitochondrial
intermembrane space, the equivalent of the bacterial periplasm. Thus, assembly of β-barrels occurs
from the same face of the membrane in bacteria and mitochondria as substrates are first imported by
the TOM complex into the intermembrane space before they engage with the SAM. This suggests that
the functional features of the eukaryotic system have been retained from the endosymbiont [33,34].
The POTRA domain of Sam50 was once reported as a signal receptor for β-barrel protein as deletion
of a short segment caused a severe phenotype and a loss in SAM-substrate interaction . The role of
POTRA in Sam50 is now thought to facilitate the folded β-barrel release into the mitochondrial outer
membrane instead of having a signal receptor role as deletion of the entire POTRA domain results
in little change from the wild type phenotype and does not affect the kinetics of β-barrel assembly
in vitro [36,37].
In addition to the central protein, Sam50, the SAM complex is also composed of two peripheral
subunits that face the cytosol, Sam35 and Sam37. Two other integral membrane proteins also
transiently act in the β-barrel folding pathway, Mdm10 and Mim1 [11,38,39]. Only Sam50 is
evolutionarily conserved from Gram-negative bacteria to mitochondria (Figure 1) [15–17]. Moreover
the peripheral subunits, Sam35 and Sam37, are actually located on the opposite side of the membrane
to the intermembrane space and hence the POTRA domain. This is in contrast to the bacterial system
where BamB to E have large domains in the periplasm. Sam35 is a receptor that binds the β-signal of
the substrate β-barrel proteins, stabilizing the substrate proteins in the SAM complex whilst strands are
being inserted into the membrane [37,40]. Sam37 promotes the release of β-barrel substrate proteins
from SAM complex at a later stage of folding . The SAM complex is not only considered to be a
β-barrel protein folding station. It is also associated with the post-folding transfer and incorporation of
proteins into their respective native complexes; it also plays a role in connections and communication
with other organelles. Hence it is considered a “hub” for membrane protein biogenesis and organelle
communication [41–43]. The metaxin1 and 2 proteins in mammalian cells are considered to perform
similar roles to Sam35 and Sam37 .
Critically, the SAM complex serves as the initial platform for assembly of the TOM complex that is
the entry portal for all imported mitochondrial proteins. The central component of the TOM complex,
the protein conducting pore, Tom40, is also a β-barrel protein. Tom40 needs to be assembled with a
series of receptor subunits including, Tom20, Tom22 and Tom70, as well as three small Tom proteins
(Tom5, Tom6, and Tom7) that seem to have a complex organising role [38,45–48].
Two other proteins which form transient associations with the SAM are Mdm10 and
Mdm12 [38,39,49]. Collectively these proteins are also important for the formation of the
endoplasmic-reticulum mitochondria encounter structure (ERMES) [38,39,41,42,49–53]. Mim1 also
interacts with the SAM complex and is important for the biogenesis of outer membrane tethered
α-helical transmembrane domain proteins [54,55].
A recent report also implicates Sam50 in the maintenance of cristae structure and respiration by
serving as a bridging point between the outer and inner mitochondrial membrane . This emphasizes the
multi-functional role of the SAM in mitochondrial biogenesis, function and maintenance. These
Int. J. Mol. Sci. 2012, 13 8042
multiple roles can be explained by the evolutionary view that eukaryotic cells developed new
mitochondrial functions based on pre-existing protein molecules as scaffolds as well as keeping the
original functions of these molecules. The role of Sam50 as a β-barrel protein insertion and folding
machine in addition to the many other roles of the SAM complex are testament to this theory.
Figure 1. Comparison of the β-barrel protein assembly machineries in Gram-negative
bacteria and eukaryotes (A) In Gram-negative bacteria, BamA is the core subunit of the
BAM complex. BamA forms a complex with lipoproteins, BamB, C, D and E in E. coli.
The BAM complex plays an important role in outer membrane biogenesis including the
insertion of autotransporters; (B) In eukaryotes, the core channel of SAM, Sam50, is the
homologue of BamA. In mitochondria, Sam50 forms a complex with the metaxins, Sam35
and Sam37. Mdm10 interacts with SAM to mediate the biogenesis of TOM. Mim1
transiently interacts with the SAM complex and is important for the biogenesis of α-helical
transmembrane domain containing proteins. BamA has five repeat POTRA domains
(P1–P5) while Sam50 only has one (P1). In eukaryotes the accessory subunits are located
on the cytosolic side of the outer membrane whilst in bacteria BamB to E are located on
the periplasmic face of the membrane. Color key-grey denotes components not conserved
between prokaryotes and eukaryotes, blue denotes conserved proteins.
5. How Functionally Conserved Are the BAM and SAM?
Since the discovery of both BamA and Sam50, many bacterial β-barrel proteins have been used to
test the ability of SAM complex to fold bacterial β-barrel proteins in mitochondria [57–60] (Table 1).
The 8-stranded monomeric barrel OmpA, the 16-stranded OmpC and PhoE from of E. coli, and
Omp85 from Neisseria meningitidis have been reported to target to mitochondria when they were
expressed in the eukaryote Saccharomyces cerevisiae. Sam50 and Sam37 were shown to be required
for PhoE assembly using genetically modified yeast strains . The C-terminal residue of PhoE,
which is required for the assembly in bacteria, is also suggested to be important for the trimeric
Int. J. Mol. Sci. 2012, 13 8043
formation of PhoE in mitochondria [57,61]. On the other hand, expression of OmpA, OmpC or PhoE
in mammalian cell lines fails to result in successful targeting and assembly in mitochondria. The
Omp85 and PorB proteins from Neisseria gonorrhoeae are recognized and inserted and folded into the
mitochondrial outer membrane by the SAM in mammalian and yeast derived mitochondria [58,60].
Furthermore, expression of a different class of bacterial β-barrel, where more than one protein subunit
is required for complete barrel formation, was also shown to be possible in mitochondria. The barrel
domain of the YadA autotransporter from Yersinia enterocolitica expressed in the yeast S. cerevisiae
showed that eukaryotes could assemble these fragments into a barrel. Trimeric YadA assembles as a
12-stranded β-barrel with a contribution of 4-strands from each YadA. The assembly process requires
the intermembrane space chaperone proteins and the SAM complex . The reasons why some and
not other bacterial proteins can be imported and folded in eukaryotic mitochondria are not known but
is likely to be related to the presence or absence of signal sequences that target them to mitochondria
and secondly be able to be recognized by the SAM.
Table 1 . Tests of bacterial β-barrel assembly in mitochondria.
Bacterial species β-barrel protein References
Bacterial β-barrels target to mitochondria during infection
Neisseria gonorrhoeae PorB [58,60,62–64]
Neisseria meningitidis PorB [60,65,66]
Acinetobacter baumannii Omp38 (AbOmpA) 
Staphylococcus aureus Panton-Valentine leukocidin (PVL) 
Bacterial β-barrels tested to be expressed in mitochondria
E. coli PhoE, OmpA, OmpC [57,58]
Yersinia enterocolitica Ya d A 
Neisseria meningitidis Omp85(BamA) 
Neisseria gonorrhoeae Omp85(BamA) 
Reciprocal experiments examining the folding and assembly of eukaryotic β-barrel proteins in
bacteria have also shown that bacterial machineries can assemble and fold eukaryotic β-barrel proteins
in the bacterial outer membrane. The eukaryotic mitochondrial porin, from Neurospora crassa, one of
the most abundant β-barrel outer mitochondrial membrane proteins of eukaryotes, can be assembled
into the bacterial outer membrane .
6. β-Barrels—Evolution and Pathogenesis
Mitochondria play dominant roles in cell death and energy metabolism . During bacterial
infection, many effector proteins are transferred to host cells for the manipulation of cell function .
It is therefore not surprising that mitochondria are targets of bacterial toxins . The fact that
bacterial β-barrels can in fact be assembled in mitochondria is not only intriguing from an evolutionary
perspective, but it is now becoming clearer. Evolutionary conserved principles are used by some
bacteria to target β-barrel toxins to host mitochondrial outer membranes via the SAM.
The bacterial outer membrane protein PorB from Neisseria gonorrhoeae (gonococcal) and
Neisseria meningitidis (meningococcal) have been found to be targeted to the mitochondria during
infection [58,60,62–66]. PorB is a β-barrel protein composed of 16 β-strands and has similar
Int. J. Mol. Sci. 2012, 13 8044
electrophysiological activity as eukaryotic porins that can be regulated by purine triphosphate
nucleotides [73,74]. The localization of PorB in mitochondria was once controversial. Meningococcal
PorB was suggested to target to the mitochondrial outer membrane while gonococcal PorB was once
reported to randomly insert into mitochondrial inner membrane [62,65]. The exact localization of PorB
in mitochondria was recently examined using a novel method combining in vitro mitochondrial import
assays and β-barrel mobility gel shift assays. PorB assembles in the mitochondrial outer membrane
using the SAM core subunit Sam50 . The molecular mechanisms behind PorB targeting to
mitochondria can be attributed to the evolutionary link between mitochondria and their bacterial
ancestors. PorB enters mitochondria through the TOM complex like all endogenous eukaryotic
β-barrels . After entry into mitochondria, the chaperone proteins in the intermembrane space, the
small Tims, are required for transport of PorB to the SAM in the outer membrane . Interestingly,
the accessory subunits of the SAM, Sam35 and Sam37, which are important for eukaryotic β-barrel
folding, are not required for PorB assembly in mitochondria . Only the core SAM subunit, Sam50,
is required. Furthermore, unlike eukaryotic β-barrels, PorB does not have a strict eukaryotic like
β-signal at the carboxy-terminus [37,60]. This could be explained by the fact that β-signal regions that
evolved in mitochondria and bacteria have diverged, with the signal in eukaryotes co-evolving with
Sam35 and Sam37. The signal regions in eukaryotes could have a greater role in the regulation of the
rate of folding but may not be absolutely required and hence are not found universally in bacteria.
Although it is generally agreed that PorB can localize to the mitochondria, its exact function remains
controversial with both pro and anti-apoptotic effects reported [62,66].
Another pathogenic β-barrel Omp38 (AbOmpA) from Acinetobacter baumannii has also been
found to target mitochondria and reported to promote cell death . AbOmpA is a bacterial outer
membrane protein which has a C-terminal OmpA-like domain which can bind peptidoglycan . The
localization of AbOmpA in mitochondria and the molecular mechanisms behind import are still
unclear but it would be anticipated that it may also use the SAM for its membrane folding/insertion.
In contrast to traditionally studied β-barrel proteins, other β-stranded bacterial proteins that are not
likely to use the SAM also exist. Panton-Valentine leukocidin (PVL) from Staphylococcus aureus has
also been identified as a mitochondrial targeted that induces apoptosis in neutrophils . PVL is a
bi-component toxin composed of two subunits, LukF-PV and LukS-PV, with the ability to form
β-barrel in membranes [76,77]. However, ability of PVL to form pores at either the mitochondrial inner
or outer membrane is yet to be demonstrated. Structural information also suggests that PVL is unlikely to
utilize the SAM as it is predicted to be structurally and functionally analogous to other leukocidin and
γ-haemolysin like toxins that firstly form multimers and a channel like structure at the membrane
surface before they insert into the membrane without assistance from other machineries .
7. Concluding Remarks and Future Directions
Hosts and pathogens co-evolve, with advantageous traits of hosts or pathogens selected for in a
given population. For bacterial pathogens this may mean that for example; a surface factor is altered to
better evade host immune defences; or a modified and more potent toxin is produced. More potent
toxins could be the result of a mutational change that alters the way a toxin interacts with its host target
protein or pathway, but it may also be the result of changing the efficiency of transport of the toxin to
Int. J. Mol. Sci. 2012, 13 8045
its target site. We propose that some pathogens, through selective forces, have continued to produce
β-barrel toxins that can be transported to mitochondria with high efficiency. As mitochondria have
diverged from their bacterial ancestors and adapted their mechanism for the import/assembly of
β-barrel outer membrane proteins to their cellular context, the bacterial pathogens that produce
β-barrel toxins that are targeted to mitochondria have by necessity also changed. Any changes in the
way the SAM recognizes β-barrel proteins, in particular if this results in changes in any signal regions
of the β-barrel substrate proteins, need to be countered by the bacterial pathogens. It will be interesting
to compare the efficiencies of import and assembly of β-barrel toxins into mammalian mitochondria
with non-toxin bacterial β-barrels on a large scale to assess for the differences as our list of bacterial
β-barrel toxins increases.
Research in the area of β-barrel protein biogenesis in Gram-negative bacteria and mitochondria
during the past decade highlights the link between the two β-barrel assembly machineries, the BAM
and SAM, respectively. The last ten years of research have revealed much about these remarkable
machines, though future research will elucidate many of the mechanistic details that are still unclear.
It is now emerging that due to their evolutionary heritage, mitochondria are targets of bacterial β-barrel
toxins. As more β-barrel bacterial toxins are discovered, the battery of substrates to probe SAM
function will increase, hopefully allowing us to understand how the exact mechanisms utilised by
BAM and SAM to insert and fold proteins in the membrane in addition to better understanding
mechanisms at play during infection.
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