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ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 277–280 doi:10.1242/jcs.137596
SHORT REPORT
A bacterial tubulovesicular network
Devrim Acehan1, Rachel Santarella-Mellwig1 and Damien P. Devos1,2,*,{
We report the presence of a membranous tubulovesicular network
in the planctomycete bacterium Gemmata obscuriglobus. This
endomembrane system interacts with membrane coat proteins
and is capable of protein internalization and degradation. Taken
together, this suggests that the planctomycetal bacterium could
illuminate the emergence of complex endomembrane systems.
KEY WORDS: Tubulovesicular network, Endomembrane system,
Planctomycetes
INTRODUCTION
The cellular space of a eukaryotic cell is highly organized
and is divided into functionally differentiated compartments
by membrane-bound structures. The origin of such complex
membranous organization is unknown and is an important issue in
cellular, molecular and evolutionary biology. Although not as
developed, bacterial intracellular organization has also proved to
be surprisingly complex. In the past few decades, membranedefined compartments have been observed in various
prokaryotes, demonstrating that cellular subfunctionalization
and differential localization also occurs in tiny organisms
(Murat et al., 2010). In this context, the planctomycete
bacterium Gemmata obscuriglobus is of considerable interest
because of its complex and dynamic endomembrane system (Lee
et al., 2009). This endomembrane system is associated with
proteins that show structural and functional similarities to the
membrane coat proteins, such as clathrin, which sustain the
eukaryotic endomembrane system (Santarella-Mellwig et al.,
2010). Furthermore, membrane internalization vesicles in G.
obscuriglobus bacteria enable the uptake and degradation of
external proteins in a process that is reminiscent of eukaryotic
endocytosis (Lonhienne et al., 2010). Other bacteria with nonclassical membrane organization, such as magnetotactic or
photosynthetic bacteria, do not display such similarities with
eukaryotes.
Historical note
Historically, the cell plan of planctomycetes has been interpreted
as being different from a classical Gram-negative (G2) one
(Fuerst, 2005; Lindsay et al., 2001). However, recent genomic
and electron-microscopy data have considerably weakened this
interpretation and instead support the hypothesis that it is a
1
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg,
Germany. 2Centre for Organismal Studies (COS), Heidelberg University,
Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
*Present address: Centro Andaluz de Biologia del Desarollo (CABD), Universidad
Pablo de Olavide, Carretera de Utrera km 1, 41013 Seville, Spain.
{
Author for correspondence ([email protected])
Received 27 June 2013; Accepted 30 October 2013
variation to the G2 cell plan (Santarella-Mellwig et al., 2013;
Speth et al., 2012; Devos, 2013). Here, we stick to the more
recent, G2-anchored, interpretation (Devos, 2013). In this
interpretation, the outermost and internal membranes are
equivalent to the G2 outer membrane and inner membrane,
respectively, defining the space between them as the periplasm.
Two cell types that are likely to represent major stages of the cell
cycle have been reported in G. obscuriglobus (Lee et al., 2009;
Santarella-Mellwig et al., 2010). The first cell type is characterized
by extensive invaginations of the inner membrane inside the
cytoplasm (Santarella-Mellwig et al., 2010; Santarella-Mellwig
et al., 2013). The second cell type has increased periplasmic
volume, which is populated by vesicle-like structures. In the course
of our analysis of the G. obscuriglobus membrane organization, we
investigated the organization of vesicles in this second cell type
using electron microscopy methods.
We previously reported the detection of proteins showing
structural and architectural similarity with the eukaryotic
membrane coat proteins sustaining the eukaryotic endomembrane
system, such as clathrin or the nucleoporins. We have shown that
these proteins are in contact with the membrane of the vesicles in the
periplasm of G. obscuriglobus cells (Santarella-Mellwig et al.,
2010). Here, we show that these vesicles sustained by the membrane
coat proteins are for the most part interconnected and form a network
of tubules and vesicles – a tubulovesicular network (TVN) – linking
the outer membrane to the inner membrane in this bacterium.
RESULTS AND DISCUSSION
Serial electron tomography of G. obscuriglobus cells resulted in
volumes where vesicle-like structures were observed in the
periplasm of the bacterial cells (Fig. 1A). We observed that some
of these vesicle-like structures were connected (Fig. 1B;
supplementary material Movie 1). A more careful investigation
revealed that most of the structures were connected to each other,
forming a continuous membrane organization within the periplasm.
This suggested the presence of a TVN, inside the periplasm of the
bacterial cells. In addition, some vesicles were connected to the
outer membrane, whereas some others were connected to the inner
membrane (Fig. 2A). Thus, the majority of the periplasmic vesicles
were connected to one another, or to one of the major cell
membranes (Fig. 2A; supplementary material Movie 1).
Volume segmentation revealed a network of connected vesicles
linked to the inner membrane and outer membrane inside the
periplasm (Fig. 2B). Thus, in these cells, the outer membrane was
connected to the inner membrane through a continuum of
membranes, forming a TVN. Such a network was found in the
periplasm of most of the cells of this type observed, representing
roughly one-third of the cells in a typical population. In addition,
these vesicles contain ribosomes, suggesting continuity with the
cytoplasm. The content of some vesicles appeared to be darker in
comparison to others or to the cytoplasm (Fig. 1B), implying
regulation of material exchange at the connections between
vesicles and subfunctionalization of the cellular space. Cellular
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Journal of Cell Science
ABSTRACT
SHORT REPORT
Journal of Cell Science (2014) 127, 277–280 doi:10.1242/jcs.137596
subfunctionalization is supported by the previous report that G.
obscuriglobus internalizes and degrades external proteins in the
periplasm (Lonhienne et al., 2010).
Immunolocalization experiments with our previously generated
antibody against one of the G. obscuriglobus membrane coat
proteins (g4978) revealed colocalization with the bacterial TVN
(Santarella-Mellwig et al., 2010). Most of the gold particles were
localized in the periplasm and a significant proportion was found in
contact with the TVN membranes (Fig. 2C). We counted 237 gold
particles in 13 cells, 74 gold particles were found associated to the
inner membrane (gold within a 15 nm distance of the membrane
center) and 125 were found in the periplasm. The ratio of the inner
membrane area to the cell area in the cross sections was found to be
0.17, giving a statistically significant P-value of 0.001 for the 31%
of gold particles associated with the inner membrane; in agreement
with our previous analysis (Santarella-Mellwig et al., 2010).
Therefore, it is likely that membrane coat-like proteins are
involved in the formation or maintenance of the bacterial TVN,
similar to their counterparts in eukaryotes.
278
These observations have implications for our interpretation and
understanding of the bacterial cell plan, and of the evolution of
compartmentalization and discrete organelle function. We report
the presence of a TVN in the periplasm of G. obscuriglobus,
which physically connects the outer membrane to the inner
membrane. This unexpected result suggests that there might be a
selection mechanism, which regulates transport between the
cytoplasm and the outside of the cell, through the periplasmic
TVN. The implications of this observation at the level of cell
biology and evolution are still to be clarified. This network
physically connects the outer membrane to the inner membrane.
This seems counterintuitive, because it appears to put the
cytoplasm in direct communication with the outside of the cell.
However, our observation of connections between vesicles of
different content demonstrates the regulation of transfer between
vesicles and thus, the filtering of transport between the outside
and the cytoplasm of the cell, through the periplasmic TVN, most
likely in both directions (export and import). This is in agreement
with the previous observation of protein internalization and
Journal of Cell Science
Fig. 1. Connected vesicles in the periplasm of the planctomycetes bacteria G. obscuriglobus. Overview of a representative cell (A) with magnified
panels of selected regions displaying the connections between vesicles (B). These regions were found at different depths within the tomogram volume, so that
the upper image is only illustrative. Arrowheads indicate connections within the TVN. Scale bar: 500 nm.
SHORT REPORT
Journal of Cell Science (2014) 127, 277–280 doi:10.1242/jcs.137596
degradation in the periplasm of those bacteria (Lonhienne et al.,
2010).
Fig. 2. A bacterial tubulovesicular network in contact with membrane coat
proteins. (A) Selected areas from different tomograms showing vesicles connected to
the outer membrane (top), to other vesicles (middle) and to the inner membrane
(bottom). C, cytoplasm; P, periplasm; IM, inner membrane; OM, outer membrane.
Arrowheads indicate selected connections between the membranes. Scale bars:
100 nm. (B) Schematic of the cellular organization of G. obscuriglobus (top left).
Periplasmic(gray), cytoplasm(white), DNA (black) and periplasmicvesicles(red) are not
shown to scale. Segmented model of the tomogram volume (middle) outer membrane
(blue), inner membrane (green) and vesicles (red) are shown on one tomogram slice.
DNA is indicated with an asterisk. Inside view of the model (bottom). Onlythe segmented
outer membrane and vesicle membranes are represented, viewed from the inside,
rotated about 90˚ from above. See supplementary material Movie 2. (C) Electron
micrographs of G. obscuriglobus cells immunolabeled with membrane coat proteins.
Arrowheads indicate selected gold particles close to the membranes of the TVN.
In the past few decades, we have learned a lot about the ancestral
eukaryotic endomembrane system, and membrane organization in
the organisms preceding the first eukaryote. Comparative
genomic and phylogenetic analyses have revealed that the first
eukaryotic cell possessed a complex endomembrane system, and
a near-modern array of the protein families associated with it
(Field and Dacks, 2009; Koumandou et al., 2013). In addition,
common aspects of function and biogenesis of functionally
distinct compartments of many eukaryotes suggest, among other
possibilities, that the primitive eukaryotic endomembrane system
might have been composed of a multifunctional TVN. This
network was probably formed by distinct communicating
compartments serving as the site of protein synthesis,
endocytosis and degradation of internalized material (Abodeely
et al., 2009). Therefore, a TVN that links the nuclear envelope to
endocytic vesicles and where degradation of the internalized
exogenous material takes place, has been suggested as a possible
characteristic feature of a primitive eukaryotic endomembrane
system (Abodeely et al., 2009). The ancestral TVN probably
communicated with membrane-bound vesicles coated with
clathrin-like membrane coat proteins at the periphery of the cell
to receive internalized material (Devos et al., 2004).
This is very similar to what we observed in G. obscuriblobus.
Here, we report the presence of a membranous TVN in this
bacterium. The presence of a TVN, together with its previously
reported features, reveals striking similarities between the G.
obscuriglobus endomembrane system and an inferred ancestral
eukaryotic one. Indeed, like the eukaryotic one, the bacterial
endomembrane system is complex and dynamic to an extent
unequalled so far in prokaryotes (Lee et al., 2009). The bacterial
inner membrane sends invaginations towards the cytoplasm that are
reminiscent of the eukaryotic endoplasmic reticulum (SantarellaMellwig et al., 2013). However, there is no nucleus-like organization
of the membranes around the DNA in the bacteria (SantarellaMellwig et al., 2013). Vesicle-like membranous structures are also
present in the bacterial periplasm (Lindsay et al., 2001). As we report
here, those vesicles are connected and form a TVN. The bacterial
endomembrane system is also involved in external compound
internalization and degradation in the periplasm, most likely through
the TVN, indicating subfunctionalization and filtering before
cytoplasmic internalization (Lonhienne et al., 2010). This feature
is unique in bacteria and related to endocytosis, which, until recently,
was held as one of the strictly eukaryotic characteristics. In addition,
this endomembrane system is in contact with proteins that are
structurally similar to membrane coat proteins, such as clathrin,
which are most likely involved in its maintenance or organization
(Santarella-Mellwig et al., 2010). There is no proof of homology
between the bacterial and eukaryotic membrane coat proteins, but
structural, architectural and functional similarities support an
evolutionary relationship between them (Devos, 2012). No sign of
lateral gene transfer in any direction, to or from eukaryotes has,
however, been detected (Santarella-Mellwig et al., 2010).
In conclusion, there is no evidence that the G. obscuriglobus
and eukaryotic endomembrane systems are related. There are
alternative explanations for the similarities observed between
the G. obscuriglobus and a putative primitive eukaryotic
endomembrane system; either planctomycetes represent an
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Journal of Cell Science
Homology between the G. obscuriglobus and the eukaryotic
endomembrane system?
SHORT REPORT
independent emergence of endomembrane organization, or the
planctomycetal endomembrane system is related to a primitive
eukaryotic one. The former could provide important information
about the formation of complex structure and convergence, whereas
the latter might provide a glimpse into the evolution of the complex
endomembrane system of modern eukaryotes. On the basis of these
similarities and others, various scenarios of relationships between
the planctomycetes and the eukaryotes have been proposed (Fuerst
and Webb, 1991; Forterre, 2011; Reynaud and Devos, 2011). In
either case, planctomycetes provide an excellent opportunity to
examine the endomembrane organization in a non-eukaryotic
system, without the complexity found in eukaryotes.
MATERIALS AND METHODS
G. obscuriglobus cells were grown as previously described (SantarellaMellwig et al., 2010). The cells were frozen with a HPM010 highpressure freezing machine (Abra Fluid, Switzerland) and freeze
substituted in an AFS2 machine (Leica, Vienna) with either 1%
osmium tetroxide, 0.1% uranyl acetate and 5% H2O and embedded in
Epon, or with 0.5% uranyl acetate and embedded in Lowicryl HM20
(Santarella-Mellwig et al., 2010). Thin (60 nm) and thick (250 nm)
sections were placed on Formvar-coated grids and 15 nm fiducial gold
markers were added. Sections were then stained with uranyl acetate and
lead citrate. Antibody labeling was carried out as previously described
(Santarella-Mellwig et al., 2010). Thin sections were imaged with a
CM120 Phillips electron microscope. For tomography, dual-tilt axis
acquisition was performed on thick sections with a Technai F30 300 kV
microscope (FEI Company). Serial sections were reconstructed and
tomograms were joined using IMOD (Kremer et al., 1996). Contours
were traced on every slice within the tomogram (about 6 nm voxel size).
To determine the statistics of antibody proximity to membranes, we
defined a membrane proximity area at a 15 nm distance from the
membrane center and counted gold particles in the membrane proximity,
in the periplasm and in the cytoplasm. We then used a one-sample
Student’s t-test with two-tailed P-value calculation using ratios of the
cell compartment area to the total cell area as expected values for a
random distribution to compare with the observed distribution of gold
particles.
Competing interests
The authors declare no competing interests.
Author contributions
Funding
D.A. and R.S.-M. are supported by European Molecular Biology Laboratory
(EMBL); D.P.P. was supported by the Centre for Organismal Studies (COS),
Heidelberg University.
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Journal of Cell Science
R.S-M. did the sample preparation, sectioning and data collection; R.S.-M. and
D.A. did the tomogram reconstruction; D.A. did the segmentation, data analysis
and the movies; D.P.D. devised and supervised the study.
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