FEMS Microbiology Letters, 362, 2015, fnv137
doi: 10.1093/femsle/fnv137
Advance Access Publication Date: 21 August 2015
Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
Biogenesis of Lysobacter sp. XL1 vesicles
Irina V. Kudryakova1,∗ , Natalia E. Suzina2 and Natalia V. Vasilyeva1
1
Laboratory of Microbial Cell Surface Biochemistry, G.K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Sciences, 5 Pr. Nauki, Pushchino, Moscow Region, 142290 Russia and
2
Laboratory of Microbial Cytology, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,
Russian Academy of Sciences, 5 Pr. Nauki, Pushchino, Moscow Region, 142290 Russia
∗ Corresponding author: Laboratory of Microbial Cell Surface Biochemistry, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,
Russian Academy of Sciences, 5 Pr. Nauki, Pushchino, Moscow Region, 142290 Russia. Tel: +7(4967)31 85 93; Fax: +7(495)956 33 70;
E-mail: [email protected]
One sentence summary: Secreted protein L5 is a factor affecting the biogenesis of Lysobacter sp. XL1 vesicles.
Editor: Klaus Hantke
ABSTRACT
The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein,
bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded
four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose
fractions were 40–65 and 65–100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction
differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be
secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic
immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space
and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation
occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained.
Keywords: Lysobacter sp. XL1; biogenesis of outer membrane vesicles; heterogeneity of outer membrane vesicles; lytic
endopeptidase L5; vesicle biogenesis factors
INTRODUCTION
Formation of outer membrane vesicles (OMVs) is a physiological feature of Gram-negative bacteria (Beveridge 1999; Kadurugamuwa and Beveridge 1997; Kuehn and Kesty 2005; Balsalobre et al. 2006; Olofsson et al. 2010; Moon et al. 2012). OMVs
are spherical structures 20–300 nm in size, whose composition
includes lipopolysaccharide, phospholipids, DNA and RNA, and
periplasmic, cytoplasmic and outer membrane proteins (Kadurugamuwa and Beveridge 1995; Horstman and Kuehn 2000;
Lee et al. 2008; Olofsson et al. 2010). OMVs play an important
role in bacterial functions: their competitive capability, acquisition of nutrients, survival under stress conditions, virulence for
pathogenic bacteria etc. (Kadurugamuwa and Beveridge 1995;
Li, Clarke and Beveridge 1996; Ciofu et al. 2000; Kobayashi et al.
2000; Renelli et al. 2004; Amano, Takeuchi and Furuta 2010; Ellis
and Kuehn 2010). Diverse information on the biogenesis of vesicles in various bacterial taxa has been accumulated (Kadurugamuwa and Beveridge 1995; McBroom and Kuehn 2007; Deatherage et al. 2009; Kulp and Kuehn 2010; Haurat et al. 2011; McMahon
et al. 2012; Lloubes et al. 2013; Schwechheimer, Kulp and Kuehn
2014). Major models are reduced to the involvement of such factors as periplasmic proteins, lipoprotein, lipopolysaccharide and
phospholipids in this process. One of the models is based on
the assumption that OMVs are formed due to the pressure of
periplasmic components (the so-called wrongly folded proteins,
peptidoglycan fragments) on the inner leaf of the bacterial outer
membrane (Schwechheimer, Kulp and Kuehn 2014). The influence of secreted extracellular proteins on vesiculation can be
Received: 18 May 2015; Accepted: 11 August 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2015, Vol. 362, No. 18
considered within the framework of this model, too. Furthermore, the involvement of OMVs as a natural transport means
for these proteins has been described in the literature (Kadurugamuwa and Beveridge 1995; Horstman and Kuehn 2000; Ciofu
et al. 2000; Kato, Kowashi and Demuth 2002; Kuehn and Kesty
2005; Amano, Takeuchi and Furuta 2010; Olofsson et al. 2010). It
is known that, using secreted proteins, bacterial cells exchange
information, provide nutrients and energy for themselves and
conquer ecological niches. It is evident that problems associated
with the understanding of protein secretion are important for
biochemistry; in this connection, their role in the biogenesis of
vesicles deserves closer consideration.
The Gram-negative bacterium Lysobacter sp. XL1 secretes bacteriolytic enzymes (peptidoglycan hydrolases) into the medium.
These enzymes break down peptidoglycan – the main structural
component of cell walls – which leads to the death of competitive bacteria. To date, five extracellular bacteriolytic enzymes
of this bacterium have been isolated and partially characterized
(Muranova et al. 2004; Stepanaia et al. 1996; Stepnaya et al. 1992,
2005; Vasilyeva et al. 2008, 2014). A broad-spectrum antimicrobial
preparation, lysoamidase, is produced based on the Lysobacter
sp. XL1 culture liquid (Kulaev et al. 2002). The most investigated
are enzymes L1 and L5, which are 60% homologous; they are also
78% and 56% homologous to α-lytic protease of Lysobacter enzymogenes (EC 3.4.21.12) (Granovsky et al. 2010, 2011; Lapteva et al.
2012). In spite of this, there are strong differences between protein L5 and its homologues in the manner of secretion into the
medium. These proteins are synthesized in the cytosol of the
bacterium as prepro proteins. Proteins of this type are known
to be secreted into the medium in two stages: first via the cytoplasmic membrane into the periplasm, which is accompanied
by the processing of its pre moiety, and then via the outer membrane, which is accompanied by the autocatalytic split-off of the
pro part and the emergence of mature protein. Enzyme L1 of
Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes,
presumably, use a secretory mechanism of type II for the second stage of secretion (Silen et al. 1989; Fujishige et al. 1992), and
protein L5, outer membrane vesicles (Vasilyeva et al. 2008, 2013).
This feature was of great interest to us. What is more, some of
our observations have enabled an assumption that protein L5
not only makes use of vesicles as a transport means, but can
also be one of their forming factors, i.e. a factor of their biogenesis. As there can be several factors that affect the biogenesis,
vesicles formed can also be heterogeneous by composition.
Lysobacter sp. XL1 vesicles of various sizes ranging from 50 to
160 nm were shown to form during the secretion of bacteriolytic
enzymes, whereas under secretion blockage conditions vesicles
formed were 20 nm in diameter (Vasilyeva et al. 2008, 2009). This
result suggests that the bacterium Lysobacter sp. XL1 forms vesicles that are heterogeneous not only in size, but also in composition and, possibly, in functions performed. Under conditions
of no secretion of bacteriolytic enzymes, Lysobacter sp. XL1 was
shown to form homogeneous vesicles. Thus, the aim of this
work was to establish the heterogeneity of Lysobacter sp. XL1
vesicles and the possible involvement of secreted protein L5 in
their formation.
and agar, 1.5%. Cultivation was done in shaken flasks containing
a 150 ml medium for 20 h (initial stage of the stationary growth
phase) with mixing at 29◦ C.
MATERIALS AND METHODS
The total protein concentration was measured by the Lowry
method (Lowry et al. 1951). The concentration of protein was calculated by the calibration curve plotted for Bovine serum albumin (Sigma, USA).
The concentration of 2-keto-3-deoxyoctanate was determined by the reaction with thiobarbituric acid (Karkhanis et al.
1978).
Bacterial strains and growth conditions
Strain Lysobacter sp. XL1 VKM B-1576 was grown in liquid and
agarized media. The liquid medium contained: peptone, 5 g L−1 ;
yeast extract, 5 g L−1 ; and NaCl, 5 g L−1 . The agarized medium
contained: peptone, 5 g L−1 ; yeast extract, 5 g L−1 ; NaCl, 5 g L−1 ;
Lysobacter sp. XL1 vesicle preparation and fractionation
in a sucrose density gradient
Cells from a 1 litre culture were removed by centrifugation at
7500 g for 20 min. Vesicles from the resulting culture liquid were
sedimented by centrifugation at 115 000 g for 2 h. The vesicle
residue was washed with 50 mM Tris/HCl buffer (pH 8.0), followed by centrifugation at the same rate; the residue was then
dissolved in 2 ml of 25% sucrose in 10 mM Tris/HCl buffer (pH
8.0) containing 5 mM EDTA. The preparation of vesicles thus
produced was layered on a stepwise sucrose density gradient
(30–55% sucrose in 10 mM Tris/HCl buffer (pH 8.0) containing 5
mM EDTA) at a step of 5% sucrose. Centrifugation was done at
106 500 g for 12 h. Fractions were collected in equal volumes,
starting from the meniscus, with account for the concentration
of sucrose. As a result, the following fractions were selected:
30%, 35%, 40% and 45% sucrose. The resulting fractions were diluted to a volume of 30 ml with 10 mM Tris/HCl buffer (pH 8.0)
and centrifuged at 115 000 g for 2 h. The residues produced were
washed in 50 mM Tris/HCl buffer (pH 8.0) at the same centrifugation rate for 1 h. The residues were resuspended in equal volumes of 50 mM Tris/HCl buffer (pH 8.0).
Electron microscopy
Negative stain
Samples were applied on the surface of a formvar film support
and stained with a 0.2% solution of uranyl acetate in water.
Electron microscopic immunocytochemistry
The immunocytochemical reaction to reveal enzyme L5 was run
on ultrathin sections of the Lysobacter sp. XL1 culture of cells
grown on an agarized medium for 20 h at 29◦ C. Cell biomass was
fixed in a 4% paraformaldehyde solution in PBS for 1 h at 4◦ C. After dehydration in a series of increasing alcohol concentrations,
material was embedded in LR White Acrylic Resin (Sigma, USA)
in accordance with manufacturer’s recommendations. The immunocytochemical reaction was run on ultrathin sections using
rabbit polyclonal antibodies to bacteriolytic enzyme L5 (dilution,
1:200) followed by treatment with protein A labelled with Au
particles of 10 nm in size (dilution, 1:100). As controls, we used
Lysobacter sp. XL1 cell sections, treated with protein A labelled
with Au (size, 10 nm), without preliminary incubation with polyclonal antibodies to enzyme L5. Upon the immunocytochemical
reaction, ultrathin sections were stained for 30 min in a 3% solution of uranyl acetate in 70% alcohol and additionally stained for
4 min with lead citrate according to Reynolds (Reynolds 1963).
Ultrathin sections and negatively stained preparations were
viewed in a JEM-100B electron microscope (JEOL, Japan) at an accelerating voltage of 80 kV.
Analytical methods
Kudryakova et al.
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Figure 1. Negative stain electron microscopy of fractions obtained in a sucrose density gradient. (A) 30% sucrose fraction. (B) 35% fraction. (C) 40% fraction. (D) 45%
fraction.
The activity of alkaline phosphatase was determined by the
rate of p-nitrophenyl phosphate hydrolysis by measuring the optical density of the mixture’s reaction at 405 nm (Torriani 1960).
The unit of phosphatase activity was taken to be the amount
of enzyme that releases one micromole of p-nitrophenol per
minute at 37◦ C.
Protein electrophoresis
Electrophoresis of proteins in 12.5% polyacrylamide gel was run
in the presence of sodium dodecyl sulfate (SDS) by the Laemmli
method (Laemmli 1970). As markers, a mixture of protein standards (Helicon, Russia) was used: cellulase, 94.6 kDa; bovine
serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; carboanhydrase,
31.0 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa. Protein
bands in gels were revealed by staining with Coomassie Brillant
Blue R-250 (Serva, Germany).
Protein immunoblotting
Enzyme immunoassay was conducted after the electrophoretic
transfer of proteins from polyacrylamide gel to the ImmunR
PVDF Membrane (Bio-Rad, USA). The transfer was run for
Blot
2 h in a 0.05 M borate buffer (pH 8.0) at cooling. The membrane
was treated with rabbit polyclonal antibodies to enzyme L5 (dilution, 1:1000), and then with the conjugate of protein A with
horseradish peroxidase at a dilution of 1:10 000 (Bio-Rad, USA).
The detection was done using SuperSignalTM West Pico chemiluminescent substrate (Pierce, USA).
RESULTS
Fractionation of a Lysobacter sp. XL1 vesicle preparation
in a sucrose density gradient
A vesicle preparation obtained from the culture liquid of Lysobacter sp. XL1 was fractionated in a 30–55% sucrose density gradient
at 106 500 g. Four fractions were obtained: of 30%, 35%, 40% and
45% sucrose. All fractions were characterized by negative stain
electron microscopy. Vesicles were found to occur in all fractions
(Fig. 1). It is seen that the 30% sucrose fraction contains vesicles
30–65 nm in diameter and occasional 70–80 nm vesicles (Fig. 1A).
Most vesicles are seen to be filled with an electron-dense content. Broken vesicles occur, with partially exposed content revealed as clumps of crystalline structure. Larger vesicles 65–100
nm in diameter, with an insignificant number of vesicles 40–50
nm in size, predominate in the 35% sucrose fraction (Fig. 1B).
This fraction also contains intact and broken vesicles. The integrity of vesicles in these fractions is supported by the activity of the periplasmic enzyme alkaline phosphatase (Table 1).
The 40% and 45% sucrose fractions contain only occasional intact vesicles 65–100 nm in diameter, along with large membrane
fragments predominant in the 40% sucrose fraction (Fig. 1C and
D, respectively).
Thus, the fractionation results confirmed the heterogeneity of Lysobacter sp. XL1 vesicles both by size and density.
Most vesicles concentrated in the 30% and 35% sucrose fractions. Vesicles of these fractions were analysed further in more
detail.
Protein and lipopolysaccharide assays of Lysobacter sp.
XL1 vesicle fractions
The 30% and 35% sucrose fractions were subjected to protein
and lipopolysaccharide assays (Table 1). It is seen from the table
that in vesicles of the 35% sucrose fraction the contents of total
protein and 2-keto-3-deoxyoctanate (Kdo) are twice as high as
compared with the 30% sucrose fraction. This can be due, on the
one hand, to the larger diameter of vesicles in the 35% fraction
as compared with those in the 30% fraction, which is consistent
with the data of electron microscopy. On the other hand, this can
be explained by a greater number of vesicles in this fraction. In
addition, an increased content of Kdo can be a consequence of
a greater amount of lipopolysaccharide in some vesicles, which
makes them heavier.
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FEMS Microbiology Letters, 2015, Vol. 362, No. 18
Table 1. Total protein content and 2-keto-3-deoxyoctanate (Kdo) concentration.
Fractions
30% sucrose fraction
35% sucrose fraction
a
Proteina (mg ml−1 )
Kdoa (mM in sample)
Alkaline phosphatase (units (mg vesicle proteins)−1 )
0.06 ± 0.01
0.17 ± 0.04
0.54 ± 0.10
1.05 ± 0.14
10.5
5.1
Values represent means ± standard deviations of double measurements performed in two independent experiments.
Electrophoretic assay of protein content in Lysobacter
sp. XL1 vesicle fractions
Enzyme immunoassay of bacteriolytic endopeptidase
L5 in fractions of Lysobacter sp. XL1 vesicles
Using SDS-PAGE, we compared the protein contents of all vesicle fractions obtained by fractionation in a sucrose density gradient (Fig. 2A). The general protein patterns of all fractions
were virtually the same. Still, noteworthy was the 30% sucrose
fraction found to have major proteins of molecular masses
24.0, 27.7, 28.7 and 54.0 kDa, which were either absent in the
other fractions or occurred in minor amounts. At the same
time, vesicles of the 30% fraction contained no major protein of
22.3 kDa found in those of all the other investigated fractions.
The proteins were analysed by MALDI-TOF mass spectrometry.
However, we failed to identify them as the proteome of Lysobacter sp. XL1 has not been completely studied, and their similarity
with proteins available in the NCBI database was low.
Thus, vesicles of the 30% sucrose fraction differed from those
of all the other fractions not only by density but also by protein
content.
An earlier study showed bacteriolytic endopeptidase L5 of
Lysobacter sp. XL1 to be secreted to the culture liquid by means
of OMVs (Vasilyeva et al. 2008). To elucidate whether protein L5
occurs in all vesicles or, alternatively, is secreted by only part
of vesicles, all vesicle fractions obtained in a sucrose density
gradient were analysed by immunoblotting with polyclonal antibodies to enzyme L5 (Fig. 2B). Analysis showed protein L5 to
be present only in vesicles of the 30% sucrose fraction, whereas
vesicles of all the other fractions did not contain it. Thus, protein
L5 was secreted only by means of vesicles occurring in the 30%
sucrose fraction, which is indicative of its selective assortment
into a certain group of vesicles. To confirm this result, we carried
out an electron microscopic immunocytochemistry of Lysobacter
sp. XL1 cells.
Electron microscopic immunocytochemistry of
Lysobacter sp. XL1 cells
Intra- and extracellular localization of enzyme L5 using the immunocytochemical reaction was carried out on ultrathin sections of Lysobacter sp. XL1 cells. Electron microscopy of the samples was done after treatment with polyclonal antibodies to
bacteriolytic enzyme L5 followed by treatment with Au-labelled
protein A. Sections of Lysobacter sp. XL1 cells treated with Aulabelled protein A, without preliminary incubation with polyclonal antibodies to enzyme L5, were used as controls to verify the non-specific binding of antibodies. In those samples, we
found no binding of antibodies (data not shown). In test samples, intercellular space was seen to comprise vesicles of two
types: containing and not containing protein L5 (Fig. 3A). Those
groups of vesicles formed within one bacterial cell (Fig. 3B). An
interesting result was that protein L5 was distributed unevenly
along the entire periplasmic space and concentrated in certain
loci of the periplasm (Fig. 3C). In addition, it is seen in Fig. 3C that
protein L5 closely adheres to the outer membrane, which can be
indicative of its affinity to it. The immunocytochemical results
show that cells of Lysobacter sp. XL1 form vesicles both containing protein L5 and not containing it, i.e. vesicles of various protein contents occur, which is consistent with the electrophoretic
and immunoblotting data.
DISCUSSION
Figure 2. (A) SDS-PAGE. A comparative electropherogram of protein content in
fractions obtained in a sucrose density gradient. The samples contained 0.04 mg
protein. The arrows point to the protein bands distinguishing the vesicles of
the 30% sucrose fraction from those of all the other fractions. The data are from
one representative of three independent experiments. (B) Immunoblotting of
vesicle fractions using polyclonal antibodies to bacteriolytic enzyme L5. The data
are from one representative of three independent experiments.
Numerous experimental data have been accumulated to date
on the biogenesis of OMVs for various taxa of Gram-negative
bacteria; the data are generalized in several models (MashburnWarren and Whiteley, 2006; Kulp and Kuehn 2010). Those data,
as well as the results of our research, indicate that all Gramnegative bacteria form vesicles by one mechanism, which proceeds as follows: the outer membrane bulges out and then
detaches to form a vesicle. The factors that determine the
process may vary, but each of them presumably affects the
Kudryakova et al.
5
Figure 3. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells using polyclonal antibodies to protein L5 and protein A conjugated with Au (size, 10
nm). (A) OMVs both containing and not containing protein L5 are seen in intercellular space. (B) heterogeneous vesicles form within one bacterial cell. (C) protein L5
concentrates in certain periplasmic loci adjacent to the inner side of the outer membrane; OM, outer membrane; P, periplasm; CM, cytoplasmic membrane; OMV + L5,
vesicles containing protein L5; OMV – L5, vesicles containing no protein L5.
rigidity of the outer membrane and, therefore, the change of its
curvature. The following three groups are considered by investigators to be the major factors. (i) Lipoprotein, which is one of the
factors that stabilize outer membrane rigidity. By its lipid moiety, it is fixed in the outer membrane; the other end is either
free or covalently bound to peptidoglycan. It has been shown
that, at the mutation of the genes of the enzymes responsible for the covalent binding of lipoprotein to peptidoglycan,
vesiculation is enhanced (Schwechheimer, Sullivan and Kuehn
2013, Schwechheimer, Kulp and Kuehn 2014). (ii) Negatively
curved molecules, which, as the result of mutual repulsion,
may disturb the rigidity of the outer membrane. Negatively
charged lipopolysaccharide may serve as one of the examples.
It has been found that the outer membrane of Pseudomonas
aeruginosa contains both neutral (A-type) and negatively charged
(B-type) lipopolysaccharides (Kadurugamuwa and Beveridge
1995). B-type lipopolysaccharide is saturated in the core part
with phosphate and carboxyl groups and consists of long negatively charged O-specific polysaccharide chains. Vesicles of P.
aeruginosa have been shown to consist of exclusively B-type
lipopolysaccharides. This is indicative of the formation of vesicles predominantly from outer membrane regions containing
this type of lipopolysaccharide, presumably as the result of electrostatic repulsion of like-charged lipopolysaccharide components (Kadurugamuwa and Beveridge 1995; Mashburn-Warren
and Whiteley, 2006). The action of acid phospholipid cardiolipin
can be attributed to the same model. It has been found to reside
in membrane loci of high curvature, which enabled an assumption of its role in the curvature process (Renner and Weibel 2011;
Lloubes et al. 2013). (iii) Periplasmic proteins, which can disturb
the rigidity of the outer membrane due to the pressure they exert
(Kulp and Kuehn 2010). Thus, mutation of the gene of Escherichia
coli periplasmic chaperone DegP led to enhanced vesiculation
due to the accumulation of wrongly folded proteins in the
periplasm (Schwechheimer, Sullivan and Kuehn 2013; Schwechheimer, Kulp and Kuehn 2014). The same effect was observed
at the mutation of the gene of permease AmpG required for secretion of muropeptides from the periplasm to the cytoplasm
for peptidoglycan recycling: large peptidoglycan fragments accumulated in the periplasm, which led to increased production
of vesicles (Schwechheimer, Sullivan and Kuehn 2013). Together
the data indicate that in one and the same bacterial cell vesicles are formed with the participation of different factors, which,
in turn, leads to the formation of heterogeneous vesicles. Some
bacteria, such as Aggregatibacter actinomycetemcomitans, E. coli
and Helicobacter pylory, have been found to form heterogeneous
vesicles (Balsalobre et al. 2006; Olofsson et al. 2010; Rompikuntal
et al. 2012).
In this work, we investigated the biogenesis of vesicles of the
Gram-negative bacterium Lysobacter sp. XL1, which, by means
of vesicles, secretes an extracellular protein, bacteriolytic endopeptidase L5. It has been shown previously that under conditions of the secretion of bacteriolytic enzymes Lysobacter sp.
XL1 forms vesicles of heterogeneous size, whereas under secretion blockage conditions vesicles formed were 20 nm in diameter (Vasilyeva et al. 2009). This result suggests that the bacterium Lysobacter sp. XL1 forms vesicles that are heterogeneous
not only with respect to size, but also composition and, possibly, performed functions. The fractionation data of a Lysobacter
sp. XL1 vesicle preparation in a sucrose density gradient confirmed this suggestion. Two fractions – of 30% and 35% sucrose –
were obtained, in which the majority of vesicles reside. An especially important result is, in our mind, associated with the finding of protein L5 only in OMVs of the 30% sucrose fraction, which
also differ from those of the 35% fraction by density and protein content. Thus, the results of our study showed that the bacterium Lysobacter sp. XL1 formed vesicles heterogeneous by size,
density and protein content. Immunocytochemistry of Lysobacter sp. XL1 cells showed that protein L5 was distributed unevenly
along the periplasmic space and concentrated in certain loci of
the periplasm from the inner side of the outer membrane. It
was in those loci that we subsequently observed a bulging of
the outer membrane. These data are in favour of the assumption that one of the possible factors affecting the formation of a
subpopulation of Lysobacter sp. XL1 vesicles containing protein
L5 can be the secreted protein itself (Fig. 4). Probably, it is the
affinity of protein L5 to the outer membrane accompanied by
its concentration in certain loci from its inner side that determines the pressure on the outer membrane, which can disturb
its rigidity. On the one hand, the mechanism of vesicle formation by Lysobacter sp. XL1 involving the secreted protein can be
attributed to the third model proposed by Kuehn et al. (Schwechheimer, Sullivan and Kuehn 2013; Schwechheimer, Kulp and
Kuehn 2014). However, in this case the process involves the secreted extracellular protein but not products unnecessary for the
cell. It is entirely possible that new data on the involvement of
secreted proteins in this process will appear. Probably, the effect
of secreted proteins can subsequently be considered as a separate model of OMV biogenesis.
In the functional aspect, the heterogeneity of vesicles with
respect to composition is of interest from the viewpoint of their
functional significance in the vital activities of the bacterial
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FEMS Microbiology Letters, 2015, Vol. 362, No. 18
Figure 4. A model of the biogenesis of Lysobacter sp. XL1 vesicles. As Lysobacter sp. XL1 vesicles are heterogeneous, they form under the influence of various factors.
One of the factors is secreted protein L5, which concentrates in certain loci of the periplasm from the inner side of the outer membrane. It is in those loci that vesicles
containing it are formed. The influence of other factors on the biogenesis of Lysobacter sp. XL1 vesicles has yet to be established. PG, peptidoglycan; P, periplasm; OM,
outer membrane; CM, cytoplasmic membrane; LPS, lipopolysaccharide; Lpp, lipoprotein; OMP, outer membrane proteins; OMV + L5, vesicles containing protein L5;
OMV – L5, vesicles containing no protein L5.
cell. Formation of inhomogeneous vesicles may allow the bacterial cell to perform several significant functions simultaneously.
Thus, for H. pylory, two subpopulations of vesicles have been
revealed, each of which has a certain set of pathogenicity factors
that can provide for the penetration of these vesicles into different host cells (Olofsson et al. 2010). Previously it has been shown
that Lysobacter sp. XL1 vesicles possess a lytic action with respect
to Gram-positive and -negative bacteria, including pathogenic
strains (Vasilyeva et al. 2014). Therefore, the role of vesicles carrying protein L5 can be reduced to the occupation of a certain
ecological niche, as well as acquisition of nutrients. The role of
the other subpopulation of Lysobacter sp. XL1 vesicles containing
no protein L5 has yet to be established.
Thus, in this study we showed that vesicles heterogeneous
by size, density and protein content formed in Lysobacter sp. XL1
within the limits of one bacterial cell. Secreted protein L5 was
found to be a possible factor affecting the biogenesis of Lysobacter sp. XL1 vesicles. The action of other factors was not ruled out.
Further studies would extend the views of this biogenesis. Subsequently, investigation of the Lysobacter sp. XL1 genome would
enable proteomic research into vesicles, which would supplement the results of this work and contribute to understanding the biogenesis of vesicles produced by Gram-negative bacteria. Additionally, the results obtained will serve as the basis for
designing artificial vesicular structures – liposomes containing
bacteriolytic enzymes – for biomedical research.
ACKNOWLEDGEMENT
We are grateful to Victor Selivanov for English translation and
Irina Tsfasman for fruitful discussions.
FUNDING
This work was supported by the Ministry of Education and Science of the Russian Federation RFMEFI60714X0013 (Agreement
No. 14.607.21.0013).
Conflict of interest. None declared.
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