Discovery of a cell wall porin in the
mycolic acid-containing actinomycete Dietzia maris
Samaneh Mafakheri1, Iván Bárcena-Uribarri1, Nafiseh Soltan Mohammadi1, Narges Abdali1,
Amanda L. Jones2, Iain C. Sutcliffe2, Roland Benz1*
1
School of Engineering and Science, Jacobs University Bremen, Campusring 1, D-28759
Bremen, Germany, and
2
Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE1
8ST, United Kingdom
*Corresponding author: Roland Benz, School of Engineering and Science
Jacobs-University Bremen, Campusring 1, D-28759 Bremen, Germany
TEL: +49 421 200-3151
FAX: +49 421 200-3249
E-mail: r.benz@jacobs-university.de
Running title: Cell wall porin of Dietzia maris
Key words: Dietzia maris, porin, lipid bilayer, cell wall
FEBS Journal format
http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1742-4658
Abstract
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The cell wall of the Gram-positive mycolic acid-containing actinomycete Dietzia maris was
found to contain a pore-forming protein, as observed from reconstitution experiments with
artificial lipid bilayer experiments in the presence of cell wall extracts. The cell wall porin was
purified to homogeneity using different biochemical methods and had an apparent molecular
mass of about 120 kDa on tricine-containing sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE). The 120 kDa protein dissociated into subunits with a molecular
mass of about 35 kDa when it was heated to 100 ºC. The 120 kDa protein formed ionpermeable channels in lipid bilayer membranes with a high single-channel conductance of
about 5.8 nS in 1 M KCl. Channel-formation was very frequent and the lifetime of the pores
was very long. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted
in an asymmetric voltage-dependence. Zero-current membrane potential measurements with
different salt solutions suggested that the porin of D. maris is cation selective because of
negative charges localized at the channel mouth. Analysis of the single-channel conductance
using non-electrolytes with known hydrodynamic radii indicated that the diameter of the cell
wall channel was about 2.1 nm. The channel characteristics of the cell wall porin of D. maris
were compared to those of other members of the mycolata. They share some common
features because they are composed of small molecular mass subunits and form large and
water-filled channels.
Introduction
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Dietzia maris was originally known as Rhodococcus maris [1] but later was classified as a
species of the new genus Dietzia. This genus has now been placed into the family
Dietziaceae within the suborder Corynebacterineae of the Actinomycetales [2,3]. Dietzia
maris is a Gram-positive, aerobic, non-spore-forming soil bacterium which forms cocci that
can develop into short rods [4]. In addition to the possibility of playing a role in waste water
treatment and its presence in activated sludge [5], it has been also reported that D. maris has
a potential application in bioremediation of soil environments [6] and as a producer of the
antioxidant canthaxanthin [7]. Dietzia sp. are only rarely reported as pathogens although they
may often be misidentified, notably as rhodococci [2,8].
D. maris belongs to the mycolata, the widespread group of mycolic acid-containing
actinomycetes [2,4,9]. These bacteria produce cell walls of a unique structure with low
permeability that contains large amounts of lipids on top of the peptidoglycan layer in form of
2-branched, 3-hydroxylated fatty acids, the mycolic acids. The mycolic acids are covalently
linked to the arabinogalactan attached to the murein of the cell wall [10,11]. There is some
evidence suggesting that this structure has a low permeability because the mycolic acids
represent a hydrophobic barrier [12-14]. This means that the mycolic acid layer has the same
function as the outer membranes of Gram-negative bacteria, which contain porins for the
passage of hydrophilic solutes. Accordingly, channel forming proteins with characteristics
similar to porins of their Gram-negative counterparts have been identified to exist in the cell
walls of some members of mycolata. They are responsible for the uptake of hydrophilic
compounds across the mycolic acid layer. Cell wall channels of different mycolata have been
characterized in recent years including Mycobacterium chelonae and Mycobacterium bovis
[15,16],
Nocardia
glutamicum,
farcinica [17,18]
Corynebacterium
Corynebacterium
amycolatum
[19-23],
diphtheriae,
Rhodococcus
Corynebacterium
equi
[24]
and
Rhodococcus erythropolis [25].
Relatively little is known about the cell envelope of D. maris beyond the presence of
arabinoglactan and relatively short mycolic acids with 34-38 carbon atoms and an apparently
truncated lipoglycan [26]. In this study, we identified a channel from the cell wall of D. maris.
A protein responsible for channel formation was identified with a molecular mass of about
120 kDa, which may be formed from a homooligomer of smaller polypeptides similar to the
situation in other members of mycolata, where the cell wall channels are frequently formed
by oligomers [18-21] According to results of the lipid bilayer conductance measurements, the
cell wall channel of D. maris is wide, water filled and has a high preference for the passage
of cations.
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Results and Discussion
Isolation and purification of the pore-forming protein from whole D. maris cells
Treatment of whole cells of representatives of the mycolata with different detergent
containing buffers has been shown to provide an efficient and simple way to isolate poreforming proteins from the cell wall [17]. The use of a number of different detergents allows
the removal of the more soluble cell wall components to obtain large amounts of the porin.
Here we used a similar method for isolation of the protein responsible for pore-forming
activity in D. maris. The cell wall proteins were extracted with different detergents such as
LDAO or Genapol X-80 and the supernatants were inspected for pore-forming activity.
Addition of the extracts to lipid bilayer membranes resulted in a very fast reconstitution of
channels and the highest pore-forming activity was present in the final supernatant
containing 1% Genapol, 10mM Tris-HCl, pH 8. SDS-PAGE of the corresponding supernatant
demonstrated that it contained a predominant protein band with an apparent molecular mass
of 120 kDa and a few other bands. Identification and further purification of the pore-forming
protein were achieved by excision of different molecular mass bands from tricine-containing
preparative SDS-PAGE gels and their extraction with 1% Genapol, 10mM Tris-HCl, pH 8
overnight at 4°C. The highest pore-forming activity was observed for the 120 kDa band (Fig.
1). To confirm whether the 120 kDa protein forms an oligomer, the protein was boiled at
100ºC for 10 min in sample buffer containing 8 M urea. SDS-PAGE of resulting sample
showed that the 120 kDa band was composed of subunits with a molecular mass of about 35
kDa (data not shown).
Interaction of the 120 kDa protein from the cell wall of D. maris with lipid bilayer membranes
We performed lipid bilayer measurements to study the interaction of the 120 kDa cell wall
protein with artificial membranes. Membranes were formed from a 1% (w/v) solution of
diphytanoyl phosphatidylcholine dissolved in n-decane. Increase of conductance was not
sudden but was observed as a function of time after the addition of the porin to the aqueous
phases on one or both sides of membranes in the black lipid state. Alternatively the porin
could be added to the aqueous phase prior to membrane formation but in this case no
defined sidedness was possible.
When different molecular mass regions were excised from the same SDS-PAGE, highest
pore-forming activity was always observed for the 120 kDa band. Addition of the pure 120
kDa protein in a very low concentration to the aqueous phase bathing black lipid membranes
resulted in a very strong increase of the conductance. Nevertheless, we also observed a
small pore-forming activity smeared across the molecular mass region between about 35 and
120 kDa. This result suggested that the 120 kDa band could be an oligomer of smaller
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polypeptides. Control experiments with the detergent Genapol at the same concentrations as
with the protein confirmed that the membrane activity was caused by the presence of the cell
wall porin and not by the detergent.
Single-channel analysis
In single-channel experiments, we observed a considerable increase of the specific
conductance of the lipid bilayers in the presence of the purified cell wall porin. The addition of
lower concentrations (10 ng/ml) of the cell wall porin to lipid bilayers made of diphytanoyl
phosphatidylcholine/n-decane allowed the resolution of stepwise conductance increases. Fig.
2A shows a single-channel recording of the D. maris 120 kDa porin activity, 5 min after the
addition of the protein to a lipid bilayer membrane. Each step indicated the incorporation of
one channel forming unit into the membrane. All the steps were directed upwards, which
demonstrated that the channels were always in the open state and had a long lifetime. Fig.
2B shows a histogram of all conductance steps observed in reconstitution experiments with
the 120 kDa protein in 1 M KCl and a voltage of 20 mV. The most frequent value for the
single-channel conductance of the 120 kDa protein was 5.75 nS.
The single-channel conductance of the pore-forming protein from D. maris was also studied
as a function of different KCl concentrations (see Table 1). The results clearly demonstrated
that the conductance was not a linear function of the KCl concentration in the aqueous phase
suggesting that the channel contained either a binding site for cations or anions or that the
channel contains point charges. Single-channel experiments were also performed with salts
other than KCl to obtain information on the size and selectivity of the channels formed by
PorADM. The results are also summarized in Table 1. The conductance sequence of the
different salts within the channel was KCl ≈ KCH3COO > LiCl. This means that the influence
of cations of different size and mobility on the conductance was quite substantial suggesting
cation-selectivity of the PorADm channel (see Table 1).
Selectivity measurements
Further information about the structure of the 120 kDa porin of D. maris was obtained from
zero-current membrane potential measurements in the presence of salt gradients. Fivefold
salt gradients (100 mM versus 500 mM) were established across lipid bilayer membranes in
which about 100 to 1000 PorADm channels were reconstituted. This gradient resulted in an
asymmetry potential of 20.7 (±2.4) mV at the more dilute side of the membrane (mean of 3
measurements). This result indicated preferential movement of potassium ions over chloride
through the channel at nearly neutral pH. The zero-current membrane potentials were
analyzed using the Goldman-Hodgkin-Katz equation [27]. The ratio of the potassium
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permeability, PK, divided by the chloride permeability, PCl, was about 6.7 (mean of 3
measurements), indicating a relatively high cation selectivity of the channel formed by
PorADm. This result was confirmed by measurements with LiCl and potassium acetate where
we observed, under the same conditions as for KCl, asymmetry potentials of 19.7 and 24.7
mV at the more dilute side, respectively for fivefold salt gradients (see Table 2). The
corresponding ratios of cation permeability Pcation divided by anion permeability Panion were 4.6
for LiCl and 7.3 for potassium acetate. This suggested that size and mobility of cations and
anions influenced the cation selectivity of PorADm, which is in agreement to the situation
observed previously for wide water-filled cell wall channels of Gram-positive bacteria, where
size and hydration shell of the ions influence selectivity [17,19,28,29]
The cell wall channel of D. maris is cation selective
Similar to channel formed by other cell wall porins such as those from M. smegmatis [30], M.
chelonae [31] and N. farcinica [32], the cell wall channel of D. maris is cation selective. This
cation selectivity is based on the presence of point charges at the channel mouth because
the single channel conductance was dependent on the square root of the cation
concentration as the data of Table 1 clearly indicated [33]. A best fit of the concentration
dependence of the single channel conductance given in Table 1 was obtained by using the
formula of Nelson and McQuarrie [34] and assuming that 2.4 negatively charged groups (q =
-3.84 x10-19 As) are located at the pore mouth and that its radius is approximately 1.1 nm.
The results of Fig. 3 show that a reasonable fit of the experimental data is achieved for these
parameters. Fig. 3 also demonstrates that the influence of the surface charges is rather small
at high ionic strength. When the single-channel conductance is corrected for the presence of
point charges at the channel mouth, it is a linear function of the aqueous salt concentration
(dashed line in Fig. 3).
The negative potential at the mouth of the channel has important implications on the function
of the cell wall channel of D. maris. At a concentration of 100 mM KCl or NaCl, the potential
is approximately -39.7 mV at the channel mouth (as calculated from the Nelson and
McQuarrie formula) [34]. This means that the concentration of monovalent cations is
increased there to 470 mM (bulk concentration 100 mM; calculated according to
c0  c  exp(   F /( R  T )) , while the concentration of monovalent anions is decreased to 21
mM (bulk concentration 100 mM; calculated according to c0  c  exp(   F /( R  T )) . This
means that under physiological conditions the channel conducts cations approximately 20times more than anions of the same aqueous mobility without being really selective for
cations due to the presence of a selectivity filter for cations. Similar considerations apply to
the discussion of the zero-current membrane potentials, i.e. only part of the full bulk aqueous
|D i e t z i a m a r i s m a n u s c r i p t
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gradient drops across the channel. It is noteworthy that also other cell wall porins form
channels that are influenced by point charges [17,21,30,31].
The channel formed by the 120 kDa protein is voltage dependent in an asymmetric manner
In single-channel recordings the PorADm channel exhibited some flickering at higher voltages,
i.e. it showed rapid transitions between open and closed configurations. This could be
caused by its voltage-dependent closing. To study this effect in more detail we increased in
multi-channel experiments the voltage across the membranes. PorADm was added at a
concentration of 100 ng/ml to one side of a black diphytanoyl phosphatidylcholine/n-decane
membrane (the trans-side) and the conductance increase was followed for about 30 minutes.
After this time the reconstitution rate of PorADm decreased significantly and we applied
different negative and positive potentials (with respect to the cis-side, the side opposite to the
addition of PorADm) to the membrane, starting from ±120 mV. Then we repeated the
experiment with ±20, ±30, ±40 up to ±100 mV. The membrane current started to decrease
when the voltage at the cis-side exceeded +20 mV. For negative potentials at the cis-side the
current was voltage-independent up to -60 mV. Only for voltages more negative than -70 mV
was a decrease was observed. The data of the voltage-dependence experiments were
analyzed in the following way: the membrane conductance (G) as a function of voltage, Vm,
was measured when the opening and closing of channels reached an equilibrium, i.e. after
the exponential decay of the membrane current following the voltage step Vm. G was divided
by the initial value of the conductance (G0, which was a linear function of the voltage)
obtained immediately after the onset of the voltage. The data in Figure 4 correspond to the
asymmetric voltage-dependence of PorADm (mean of three membranes) when the protein
was added to the trans-side. This result indicated full asymmetric insertion of PorADm into the
membranes when it was added to only one side. The addition of the protein to both sides of
the membrane resulted in a symmetric response to the applied voltage (see Figure 4).
Estimation of the diameter of the cell wall porin of D. maris
Experiments with non-electrolytes (NEs) can be used to provide an estimation of the effective
pore diameter of a channel [35-41]. This method was also applied to the conductance of the
PorADm channel. The 1 M KCl solution was supplemented for these experiments with 20%
(w/v) NEs with different molecular masses ranging from 62 Da (ethylene glycol) to 3 kDa
(PEG 3000). This means that the NEs had molecular radii between 0.26 nm to 1.44 nm (see
Table 3). The results of single-channel conductance measurements with these NEs
suggested that large NEs with hydrodynamic radii between 1.22 and 1.44 nm (PEG 2000
and PEG 3000) did not enter the PorADm channel and showed only little or no effect on its
|D i e t z i a m a r i s m a n u s c r i p t
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conductance. However, in the presence of small NEs with hydrodynamic radii up to 0.80 nm,
the single-channel conductance of PorADm decreased approximately proportional to that of
the bulk aqueous conductivity (Table 3). Histograms of four representative NEs
measurements together with the recordings of single-channel traces are illustrated in Figure
5. For PEG 1000, (hydrodynamic radius 0.94 nm) the single-channel conductance decreased
but notably much less than that of the specific conductivity of the aqueous bulk solution (see
Table 3 and Fig. 5). These data indicated only partial filling of the PorADm channel with the
corresponding NE [35] suggesting that non-electrolytes with a much larger hydrodynamic
radius than 0.94 nm were not able to enter the cell wall channel, thus showing no reduction
in the pore conductance. In contrast, non-electrolytes with a hydrodynamic radius of 0.8 nm
or smaller could enter and presumably also pass through the channel (Table 3). To
characterize the pore size of PorADm in more detail, its conductance dependent on the
different NEs was plotted as a function of their molecular masses and hydrodynamic radii
following established procedures [42]. The ratios of the single-channel conductance in the
presence of NEs to that in the absence of NEs as a function of the molecular mass and the
NEs radii are shown in Fig.6. The obtained results suggested that NEs with a mean
molecular mass (Mr) of ≤ 600 g/mol and a hydrodynamic radius (r) ≤ 0.8 nm enter fully the
channel whereas NEs with a Mr ≥ 2000 g/mol and r ≥ 1.22 nm cannot enter the PorADm
channel. The results of these measurements suggested that the entrance diameter of PorADm
is around 2.1 nm.
Comparison of the cell wall channel properties of D. maris and other mycolata
The cell envelope of D. maris and members of the closely related genera Corynebacterium,
Mycobacterium, Nocardia and Rhodococcus are similar in the composition and organization
of the cell wall, with an asymmetric outer lipid layer defined by the presence of mycolic acids
[10,11]. Therefore, it is expected that the porins in the cell wall of these bacteria share some
common properties (Table 4). These include that they are typically composed of small
molecular mass subunits and form large and water-filled channels. These common
properties seem to be a characteristic for the cell wall porins of mycolata. However, some
exhibit cation selectivity (as in D. maris) whereas others are anion selective, although it is
likely that each organism will possess channels with both types of selectivity in order to
facilitate uptake of the full range of hydrophilic solutes. More interestingly, some porins
appear to work as hetero-oligomers [47,48] whereas others form likely homo-oligomers [49] .
Materials and Methods
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Bacterial strain and growth conditions
D. maris DSM 43672T cells were grown in GYE Glucose-Yeast Extract medium (10 g
Glucose and 10 g Yeast Extract per L distilled water), pH 7.2 at 28 ºC with shaking. Cells
were checked for purity and harvested by centrifugation at early stationary phase. The cells
were washed twice with sterile distilled water and lyophilized.
Isolation and purification of the channel-forming protein from the cell wall
For purification of the cell wall porin, we used the same method used for the extraction of the
cell wall channel of N. farcinica [17]. The method is based on the use of different detergents
to remove most of the soluble cell wall molecules. In the first two steps (I and II), the
lyophilized cells were washed in 10mM Tris-HCl, pH 8.0 followed by centrifugation at 4000
rpm in an Eppendorf 5810R centrifuge (rotor A-4-81) for 10 min. The pellet was then treated
with two different washing steps (steps III and IV) using 0.2 % SDS, 1% Genapol and 10 mM
EDTA followed by centrifugation (11,000 rpm in an Eppendorf 5415R microcentrifuge, 4°C)
the final pellet was homogenized in 1% Genapol and 10 mM Tris-HCl, pH 8 for 20 h at 50ºC
under agitation. Pore-forming activity was present in the resulting pellet and in its
supernatant.
SDS-PAGE
Analytical and preparative SDS-PAGE was performed according to Laemmli [50]. Because of
the low resolution of this gel system for observation of low molecular mass proteins, tricine
containing gels were used as described by Schägger [51]. The gels were stained with
Coomassie brilliant blue, Colloidal Coomassie blue [52] or with silver stain [53].
Lipid bilayer experiments
The methods used for the lipid bilayer measurements have been described previously in
detail [54]. A Teflon chamber was divided into two compartments connected by a small
circular hole with a surface area of about 0.4 mm2. Black lipid bilayer membranes were
obtained by painting a solution of 1% (w/v) diphytanoyl phosphatidylcholine in n-decane onto
the hole. All salts were analytical grade and the temperature was maintained at 20°C during
all experiments. Zero current membrane potential measurements were obtained by
establishing a salt gradient across membranes containing about 100 cell wall porins as
described elsewhere [27].
Estimation of the channel diameter using non-electrolytes (NEs)
The method used was as described by Krasilnikov et al. in 1998 [35]. Solutions containing
1M KCl were supplemented with 20% (w/v) of an appropriate NE. Single-channel
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measurements were carried out using different NE with increasing hydrodynamic radius. The
conductance in the presence of the NEs was calculated from at least 100 single events.
Acknowledgements
References
[1] Nesterenko OA, Nogina TM, Kasumova SA, Kvasnikov EI & Batrakov SG (1982)
Rhodococcus luteus nom. nov. and Rhodococcus maris nom. Nov. Int J Syst
Bacteriol 32, 1-14.
[2] Keorner RJ, Goodfellow M & Jones AL (2009) The genus Dietzia: a new home for
some known and emerging opportunist pathogens. FEMS Immunol Med Microbiol 55,
296-305.
[3] Zhi XY, Li WJ & Stackebrandt E (2009) An update of the structure and 16S rRNA
gene sequence-based definition of higher ranks of the class Actinobacteria, with the
proposal of two new suborders and four new families and emended descriptions of
the existing higher taxa. Int J Syst Evol Microbiol 59, 589-608.
[4] Rainey FA, Klatte S, Kroppenstedt RM & Stackebrandt E (1995) Dietzia, a New
Genus Including Dietzia maris comb. nov. Formerly Rhodococcus maris. Int J Syst
Bacteriol 45, 32-36.
[5] Schuppler M, Wagner M, Schön G & Göbel UB (1998) In situ identification of
nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted
oligonucleotide Probes. Microbiology 144, 249-259.
[6] Lima TMS, Fonseca AF, Leao BA, Mounteer AH, Totola MR & Borges AC (2011) Oil
Recovery from Fuel Oil Storage Tank Sludge Using Biosurfactants. J Bioremed
Biodegrad 2, 125. doi:10.4172/2155-6199.1000125.x
[7] Goswami G, Chakraborty S, Chaudhuri S & Dutta D (2012) Optimization of process
parameters by response surface methodology and kinetic modeling for batch
production of canthaxanthin by Dietzia maris NIT-D (accession number: HM151403).
Bioprocess Biosyst Eng 35,1375–1388.
[8] Niwa H, Lasker BA, Hinrikson HP, Franzen CG, Steigerwalt AG, Whitney AM & Brown
JM (2012) Characterization of human clinical isolates of Dietzia species previously
misidentified as Rhodococcus equi. Eur J Clin Microbiol Infect Dis 31, 811–820.
[9] Nishiuchi Y, Baba T & Yano I (2000) Mycolic acids from Rhodococcus Gordonia and
Dietzia. J Microbiol Methods 40, 1-9.
[10] Sutcliffe IC, Brown A & Dover L (2010) The rhodococcal cell envelope: composition,
organisation and biosynthesis. In Biology of Rhodococcus (Alvarez HM, eds), pp. 2971. Springer Berlin Heidelberg.
[11] Brennan P & Nikaido H (1995) The envelope of Mycobacteria. Annu Rev Biochem 64,
29-63.
|D i e t z i a m a r i s m a n u s c r i p t
10
[12] Jarlieri V & Nikaido H (1990) Permeability Barrier to Hydrophilic Solutes in
Mycobacterium chelonei. J Bacteriol 172, 1418-1423.
[13] Gordon HJN & Kajioka R (1977) Permeability barrier to rifampin in mycobacteria.
Antimicrob. Agents Chemother 11, 773-779.
[14] David HL, Rastogi N, Clavel-Seres S, Clement F & Thorel MF (1987) Structure of the
cell envelope of Mycobacterium avium. Zentralbl Bakteriol Mikrobiol Hyg Ser A 264,
49-66.
[15] Trias J, Jarlier V & Benz R (1992) Porins in the cell wall of mycobacteria. Science
258, 1479–1481.
[16] Lichtinger T, Heym B, Maier E, Eichner H, Cole ST & Benz R (1999) Evidence for a
small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a
wide cation-selective pore. FEBS Lett 454, 349-355.
[17] Rieß FG, Lichtinger T, Cseh R, Yassin AF, Schaal KP & Benz R (1998) The cell wall
channel of Nocardia farcinica: biochemical identification of the channel-forming
protein and biophysical characterization of the channel properties. Mol
Microbiol 29,139-150.
[18] Kläckta C, Knörzer P, Rieß F & Benz R (2011) Hetero-oligomeric cell wall channels
(porins) of Nocardia farcinica. Biochim et Biophys Acta 1808, 1601–1610
[19] Schiffler B, Barth E, Daffé M & Benz R (2007) Corynebacterium diphtheriae:
Identification and characterization of a channel-forming protein in the cell wall. J
Bacteriol 189, 7709-7719.
[20] Niederweis M, Maier E, Lichtinger T, Benz R & Krämer R (1995) Identification of
channel-forming activity in the cell wall of Corynebacterium glutamicum. J
Bacteriol 177, 5716-5718.
[21] Lichtinger T, Burkovski A, Niederweis M, Krämer R & Benz R (1998) Biochemical and
biophysical characterization of the cell wall channel of Corynebacterium glutamicum:
the channel is formed by a low molecular mass subunit. Biochemistry 37, 15024–
15032.
[22] Dörner U, Schiffler B, Lanéelle MA, Daffé M & Benz R (2009) Identification of a cellwall channel in the corynemycolic acid-free Gram-positive bacterium
Corynebacterium amycolatum. Int Microbiol 12, 29-38.
|D i e t z i a m a r i s m a n u s c r i p t
11
[23] Soltan Mohammadi N, Mafakheri S, Abdali N, Barcena-Uribarri I, Tauch A & Benz R
(2013) Identification and characterization of the channel-forming protein in the cell
wall of Corynebacterium amycolatum. Biochim Biophys Acta 1828, 2574–2582.
[24] Rieß FG, Elflein M, Benk M, Schiffler B, Benz R, Garton N& Sutcliffe I (2003) The Cell
Wall of the Pathogenic Bacterium Rhodococcus equi Contains Two Channel-Forming
Proteins with Different Properties. J Bacteriol 185, 952–2960.
[25] Lichtinger T, Reiss G & Benz R (2000) Biochemical identification and biophysical
characterization of a channel-forming protein from Rhodococcus erythropolis. J
Bacteriol 182, 764-770.
[26] Sutcliffe IC (2000) Characterisation of a lipomannan lipoglycan from the mycolic acid
containing actinomycete Dietzia maris. Antonie Van Leeuwenhoek. 78:195-201.
[27] Benz R, Janko K & Lauger P (1979) Ionic selectivity of pores formed by the matrix
protein (porin) of Escherichia coli. Biochim Biophys Acta 551, 238-47.
[28] Rieß FG, Dörner U, Schiffler B & Benz R (2001) Study of the properties of a channelforming protein of the cell wall of the gram-positive bacterium Mycobacterium phlei. J
Membrane Biol 182, 147-157.
[29] Hunten P, Schiffler B, Lottspeich F & Benz R (2005) PorH, a new channel-forming
protein present in the cell wall of Corynebacterium efficiens and Corynebacterium
callunae. Microbiology 151, 2429-2438.
[30] Trias J & Benz R (1994) Permeability of the cell wall of Mycobacterium smegmatis.
Mol Microbiol 14, 283-290.
[31] Trias J & Benz R (1993) Characterization of the channel formed by the mycobacterial
porin in lipid bilayer membranes. Demonstration of voltage gating and of negative
point charges at the channel mouth. J Biol Chem 268, 6234-6240.
[32] Riess FG, Lichtinger T, Cseh R, Yassin AF, Schaal KP & Benz R (1998) The cell wall
porin of Nocardia farcinica: biochemical identification of the channel-forming protein
and biophysical characterization of the channel properties. Mol Microbiol 29, 139-150
[33] Maier E, Reinhard N & Benz R (1996) Channel-forming activity and channel size of
the RTX toxins ApxI, ApxII, and ApxIII of Actinobacillus pleuropneumoniae. Infect
Immun 64, 4415-4423.
[34] Nelson AP & McQuarrie DA (1975) The effect of discrete charges on the electrical
properties of a membrane. J Theor Biol 55, 13-27
[35] Krasilnikov OV, Da Cruz JB, Yuldasheva LN, Varanda WA & Nogueira RA (1998) A
novel approach to study the geometry of the water lumen of ion channels: colicin Ia
channels in planar lipid bilayers. J Membr Biol 161, 83-92.
|D i e t z i a m a r i s m a n u s c r i p t
12
[36] Rostovtseva TK, Nestorovich EM & Bezrukov SM (2002) Partitioning of differently
sized poly(ethylene glycol)s into OmpF porin. Biophys J 82, 160-169.
[37] Berestovsky GN, Ternovsky VI & Kataev AA (2001) Through pore diameter in the cell
wall of Chara corallina. J Exp Bot 52, 1173-1177.
[38] Ternovsky YI, Okada Y & Sabirov RZ (2004) Sizing the pore of the volume-sensitive
anion channel by differential polymer partitioning. FEBS Lett 576, 433-436.
[39] Vodyanoy I & Bezrukov SM (1992) Sizing of an ion pore by access resistance
measurements. Biophys J, 62, 10-11.
[40] Sabirov RZ, Krasilnikov OV, Ternovsky VI & Merzliak PG (1993) Relation between
ionic channel conductance and conductivity of media containing different
nonelectrolytes. A novel method of pore size determination. Gen Physiol Biophys 12,
95-111.
[41] Krasilnikov OV, Sabirov RZ, Ternovsky VI, Merzliak PG & Muratkhodjaev JN (1992)
A simple method for the determination of the pore radius of ion channels in planar
lipid bilayer membranes. FEMS Microbiol Immunol 5, 93-100.
[42] Krasilnikov OV (2002) Sizing channels with neutral polymers. In Structure and
Dynamics of Confined Polymers (Kasianowicz J, Kellermayer MZ and Deamer D
eds), pp. 97-115. NATO Science Series, Springer Netherlands.
[43] Rieß FG, Lichtinger T, Yassin AF, Schaal KP & Benz R (1999) The cell wall porin of
the gram-positive bacterium Nocardia asteroides forms cation-selective channels that
exhibit asymmetric voltage-dependence. Arch Microbiol 171, 173-182.
[44] Rieß FG & Benz R (2000) Discovery of a novel channel-forming protein in the cell wall
of the non-pathogenic Nocardia corynebacteroides. Biochim Biophys Acta 1509, 485495.
[45] Niederweis M, Ehrt S, Heinz C, Klöcker U, Karosi S, Swiderek KM, Riley LW & Benz
R (1999) Cloning of the mspA gene encoding a porin from Mycobacterium
smegmatis. Mol Microbiol 33, 933-945.
[46] Trias J and Benz R (1994) Permeability of the cell wall of Mycobacterium smegmatis.
Mol Microbiol 14, 283-290.
[47] Barth E, Barcelo MA, Klackta C & Benz R (2010) Reconstitution experiments and
gene deletions reveal the existence of two-component major cell wall channels in the
genus Corynebacterium. J Bacteriol 192, 786-800.
|D i e t z i a m a r i s m a n u s c r i p t
13
[48] Klackta C, Knorzer P, Riess F & Benz R (2011) Hetero-oligomeric cell wall channels
(porins) of Nocardia farcinica. Biochim Biophys Acta 1808, 1601-1610.
[49] Abdali N, Barth E, Norouzy A, Schulz R, Nau WM, Kleinekathöfer U, Tauch A & Benz
R (2013) Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long
PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel.
PLoS One 8, 1-17.
[50] Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227, 680-685.
[51] Schägger H (2006) Tricine–SDS-PAGE. Nature protocols 1, 16-22.
[52] Erhardt W, Neuhoff V, Arnold N & Taube D (1988) Improved staining of proteins in
polyacrylamide gels including isoelectric focusing gels with clear background at
nanogram sensitivity using CBB G-250 and R-250. Electrophoresis 9, 255-262.
[53] Gross HJ, Baier H & Blum H (1987) Improved silver staining of plant proteins, RNA
and DNA in polyacrylamide gels. Electrophoresis 8, 93-99.
[54] Benz R, Janko K, Boos W & Laüger P (1978) Formation of large, ion-permeable
membrane channels by the matrix protein (porin) of Escherichia coli. Biochim Biophys
Acta 511, 305-319.
|D i e t z i a m a r i s m a n u s c r i p t
14
Tables
Table 1. Average single-channel conductance of D. maris pore-forming protein in
different electrolyte solutions.
Electrolyte
Concentration (M)
G (nS)
KCl
0.01
0.5 ± 0.1
0.03
0.75 ± 0.2
0.1
1.5 ± 0.5
0.3
2.5 ± 0.5
1
5.75 ± 0.5
3
12.5 ± 2.5
LiCl
1
2.5 ± 0.5
KCH3COO (pH 7)
1
4 ± 0.5
The membranes were formed by diphytanoyl phosphatidylcholine/ in n-decane. The singlechannel conductance was measured at 20mV applied voltage and T = 20°C. The average
single-channel conductance, G (± SD), was calculated from at least 100 single events.
|D i e t z i a m a r i s m a n u s c r i p t
15
Table 2. Zero-current membrane potentials, Vm, of PC/n-decane membranes in the presence
of PorA of D. maris measured for a 5-fold gradient of different saltsa.
Electrolyte
Permeability ratios
Vm (mV)
Pcation/Panion
KCl
6.7
20.7 ± 2.4
LiCl
4.6
19.7 ± 1.7
KCH3COO (pH 7)
7.3
24.7 ± 2.3
aVm is defined as the difference between the potential at the dilute side minus the potential at
the concentrated side. The aqueous salt solutions were used unbuffered and had a pH of 6,
if not indicated otherwise; T = 20°C. The permeability ratio Pcation/Panion was calculated using
the Goldman-Hodgkin-Katz equation [27] as the mean of at least 3 individual experiments.
|D i e t z i a m a r i s m a n u s c r i p t
16
Table 3. Estimation of the diameter of the cell wall porin of D. maris.
Mr (g/mol)
r (nm)
G (nS)
G(+NE)/(G-NE)
X (mS cm-1)
-
-
5.75 ± 0.5
1
110.3
Ethylene glycol
62
0.26
3.25± 0.25
0.57 ± 0.09
57.2
Glycerol
92
0.31
2.50± 0.25
0.43 ± 0.08
49.1
Arabinose
150
0.34
2.75± 0.25
0.48 ± 0.09
63.7
Sorbitol
182
0.39
3.00± 0.25
0.52 ± 0.08
57.8
PEG 300
300
0.60
2.75± 0.25
0.48 ± 0.09
45.5
PEG 400
400
0.70
2.75± 0.50
0.48 ± 0.10
46.4
PEG 600
600
0.80
2.75± 0.25
0.48 ± 0.08
54.1
PEG 1000
1000
0.94
4.75± 0.25
0.83 ± 0.10
49.5
PEG 2000
2000
1.22
5.00± 0.25
0.87 ± 0.11
43.6
PEG 3000
3000
1.44
5.00± 0.25
0.87 ± 0.10
48.9
Non-electrolyte
None
The aqueous phase contained 1 M KCl and 20% (w/v) of different non-electrolytes. The nonelectrolytes were used to determine the diameter of the 120 kDa porin. The molecular
masses and the radii (r) of the non-electrolytes are given together with the conductance of
the channel (G ± SD) in the corresponding solution and the conductivity of the solution [40].
The membrane voltage was 20 mV; T = 20°C.
|D i e t z i a m a r i s m a n u s c r i p t
17
Table 4. Comparison of the properties of cell wall channels of different mycolata.
Cell wall porin
G (nS)
MW
(kDa)
Selectivity
Voltage
dependenc
e
yes
Channel
diameter
(nm)
2.2
Refs.
Corynebacterium
diphtheriae
CdPorA_ATCC
11913
CdPorH_ATCC
11913
Corynebacterium
glutamicum
CgPorA ATCC
13032
CgPorH ATCC
13032
Corynebacterium
amycolatum.
Corynebacterium
callunae
hyp_CcPorA_ATC
C 15991
CcPorH_ATCC
15991
Corynebacterium
efficiens
CefPorH
CefPorA
Nocardia farcinica
NfpA
NfpB
Nocardia
asteroides
Nocardia
corynebacteroides
Mycobacterium
bovis
Mycobacterium
smegmatis
Mycobacterium
chelonae
Mycobacterium
phlei
Rhodococcus
erythropolis
Rhodococcus equi
PorA
PorB
Dietzia maris
2.25
66
small selectivity
for cations
cation selective
-
2.2
[20][21]
cation selective
yes
cation selective
yes
small selectivity
for anions
yes
cation selective
yes
cation selective
yes
[19]
4.7
6.8
5.5
2.5
4.7
6.2
3.8
45
3.0±0.4
[22][23]
2.2
[29]
4.8
6
2.3
[29]
3
6
4
87
31
30
84
5.5
134
cation selective
yes
100
cation selective
anion selective
cation selective
Yes
no
yes
3.0
[45][46]
2.7
59
cation selective
yes
2.2
[15]
4.5
135
cation selective
yes
1.8 to 2.0
[28]
6
67
cation selective
yes
2.0
[25]
4
0.3
5.75
67
11
120
cation selective
anion selective
cation selective
Yes
No
yes
2.0
[24]
0.9
This
study
3
4
0.78
|D i e t z i a m a r i s m a n u s c r i p t
1.4-1.6
[17][18]
[43]
1.0
[44]
[16]
18
Figure legends
Figure 1. 10% SDS-PAGE of final supernatant and purified cell wall porin. Lane 1:
Molecular mass markers 170 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa and
25 kDa. Lane 2: The final supernatant. Lane 3: The pure cell wall porin of D. maris was
obtained by elution of the 120 kDa band from tricine-containing preparative SDS-PAGE.
Figure 2.
(A) Single-channel recording of a PC/n-decane membrane in presence of pure 120 kDa
protein of D. maris. The aqueous phase contained 1 M KCl, 10 mM Tris-HCl (pH 8.0), the
applied membrane potential was 20 mV and the temperature was 20°C.
(B) Histogram of the probability P(G) for the occurrence of a given conductivity unit in
PC/n-decane membranes in the presence of the 120 kDa protein of D. maris. The most
frequent single-channel conductance (G/nS) was 5.75 nS for a total of about 147 singlechannel events. The data were collected from different individual membranes. Aqueous
phase contained 1 M KCl, the applied voltage was 20 mV and the temperature was 20 °C.
Figure 3. Single-channel conductance of the cell wall channel of D. maris as a function
the KCl concentration in the aqueous phase (filled squares). The solid line represents the
fit of the single-channel conductance data with the Nelson and McQuarrie (1975) formulas
(eqs. (1) to (3) of Benz and Trias, 1993) assuming the presence of negative point charges
(2.4 negative charges; q = -3.84 x 10-19 As) at the channel mouth and assuming a channel
diameter of 2.2 nm. c, concentration of the KCl solution in M (molar); G, average singlechannel conductance in nS (nano Siemens, 10-9 S). The broken (straight) line shows the
single-channel conductance of the cell wall channel that would be expected without point
charges. It corresponds to a linear function between channel conductance and bulk aqueous
concentration.
Figure 4. Voltage dependence of D. maris 120 kDa protein when the porin was added
to one or both sides of the membrane. The ratio of the conductance (G) at an applied
voltage (V), divided by the initial value of the conductance (G0), immediately after the turning
on of the voltage. The aqueous phase contained 1 M KCl and 100 ng/ml porin. The
membranes were formed from diphytanoyl phosphatidylcholine /n-decane at a temperature
of 20°C.
Figure 5. Single-channel recording and histogram of PorADM in the presence of
different non-electrolytes. Ethylene glycol and arabinose with hydrodynamic radii of 0.26
and 0.34 nm can get inside the porin showing reduction in the conductance while PEG 1000
and PEG 2000 apparently can not enter the channel.
Figure 6. Dependence of the single-channel conductance, G, of PorADm on the
molecular masses and radii of the non-electrolytes (NEs). G(+NE)/G(-NE) (± SD) is the ratio
of the mean single-channel channel conductance in the presence of NEs divided by that in
the absence of NEs (5.75 nS) at pH 6. Molecular masses and radii of the non-electrolytes
were taken from Table 1.
|D i e t z i a m a r i s m a n u s c r i p t
19
Figures
1
2
3
130
Fig. 1.
|D i e t z i a m a r i s m a n u s c r i p t
20
Fig. 7.
Fig. 2.
|D i e t z i a m a r i s m a n u s c r i p t
21
Fig. 3
|D i e t z i a m a r i s m a n u s c r i p t
22
Fig. 4.
|D i e t z i a m a r i s m a n u s c r i p t
23
Arabinose
Ethylene Glycole
3 nS
3 nS
5s
5s
PEG2000
PEG1000
4 nS
5s
5 nS
5s
Fig. 6.
|D i e t z i a m a r i s m a n u s c r i p t
24
Fig. 5.
|D i e t z i a m a r i s m a n u s c r i p t
25
Fig. 6.
|D i e t z i a m a r i s m a n u s c r i p t
26