1 Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
2 Max-Planck-Institute for Biochemistry, Department for Protein Analytics, Am Klopferspitz 18A, D-82152 Martinsried, Germany
Correspondence
Roland Benz
roland.benz{at}mail.uni-wuerzburg.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ871585 and AJ871586.
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INTRODUCTION |
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C. efficiens, C. callunae and C. glutamicum share similar cell-wall composition. The cell wall of mycolata contains a second layer consisting of covalently bound mycolic acids and extractable lipids (Barksdale, 1981; Goodfellow et al., 1976
; Minnikin, 1987
; Ochi, 1995
) in addition to the thick peptidoglycan layer. The mycolic acids are 2-branched, 3-hydroxylated fatty acids and variable in chain length. The cell wall of corynebacteria contains mycolic acids with a chain length of about 2238 carbon atoms, whereas other members of the mycolata contain much longer mycolic acids (Minnikin, 1987
; Yano & Saito, 1972
; Minnikin et al., 1974
; Daffé et al., 1990
; Holt et al., 1994
; Brennan & Nikaido, 1995
). The mycolic acid layer represents a permeability barrier (Liu et al., 1995
, 1996
; Nikaido et al., 1993
), similar to the outer membrane of Gram-negative bacteria. To overcome this barrier, channel-forming proteins, so-called porins, are necessary to allow the passage of hydrophilic solutes. With respect to the transport of amino acids over this barrier, it is of particular importance to characterize the hydrophilic pathways in the cell wall of corynebacteria.
The first porin identified in the cell wall of a member of the mycolata consisted of a 59 kDa cell-wall protein with a mean single-channel conductance of 2·7 nS in 1 M KCl from Mycobacterium chelonae (Trias et al., 1992; Trias & Benz, 1993
). For this bacterium it has been demonstrated that the permeability of the cell wall for hydrophilic solutes is slightly lower than that of Pseudomonas aeruginosa and much lower than that of Escherichia coli (Jarlier & Nikaido, 1990
; Trias & Benz, 1993
). This could explain why members of the mycolata have a low susceptibility towards certain antibiotics. Since the discovery of the first cell-wall channel, several porins have been identified and characterized in members of the mycolata (Riess et al., 1998
; Lichtinger et al., 1998
, 1999
, 2000
, 2001
; Costa-Riu et al., 2003b
). Common to most of them is the formation of wide and water-filled pores that are cation-selective due to negative charges (Trias & Benz, 1993
, 1994
; Riess et al., 1998
; Lichtinger et al., 1998
, 1999
, 2001
).
PorA from C. glutamicum was the first pore-forming protein of the corynebacteria that was characterized. The channel is cation-selective with a single-channel conductance of about 5·5 nS in 1 M KCl and it is formed by an oligomer of a small 45 aa polypeptide that is encoded without a leader sequence (Lichtinger et al., 1998, 2001
). Another porin, PorB, of 99 aa, was found after deleting the porA gene from the C. glutamicum chromosome (Costa-Riu et al., 2003a
, b
). PorB forms anion-selective channels with a single-channel conductance of about 700 pS in 1 M KCl in lipid-bilayer experiments. To extend the knowledge of channel-forming proteins of corynebacteria, we screened the cell walls of two closely related coryneform species, C. callunae and C. efficiens, for channel-forming proteins. Two homologous cell-wall channel proteins, PorHC.call and PorHC.eff, were identified in the two species. The former is highly cation-selective, whereas the latter is slightly anion-selective. Both are voltage-dependent and their single-channel conductances are similar in 1 M KCl. The channel-forming proteins were purified to homogeneity and their biophysical properties were studied in detail. The proteins were partially sequenced and a sequence alignment search within the known chromosome of C. efficiens demonstrated that it contained a gene that matched the partial amino acid sequence of PorHC.eff.
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METHODS |
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Isolation and purification of channel-forming proteins.
For the isolation of the channel-forming proteins from C. efficiens AJ 12310 and C. callunae ATCC 15991, a method was used that has been described previously for the isolation and purification of PorAC.glut of C. glutamicum (Lichtinger et al., 1999). It is based on the extraction of whole cells with organic solvents and avoids the substantial loss of material caused by sucrose density centrifugation of the cell envelope to separate the cytoplasmic membrane from the cell-wall fraction. For the extraction procedure 200 ml cells was grown to an OD600 of 10 and harvested by centrifugation (10 000 r.p.m. for 10 min in a Beckman J2-21M/E centrifuge). The cells were washed twice in 10 mM Tris/HCl (pH 8). The washed and centrifuged cells were extracted twice with organic solvent, a 1 : 2 mixture of chloroform/methanol in a proportion of 1 part cells and 58 parts chloroform/methanol. The duration of the extraction was about 3 h at room temperature with stirring in a closed tube to avoid loss of chloroform. Cells and the chloroform/methanol solution were centrifuged for 15 min (10 000 r.p.m. in a Beckman J2-21M/E centrifuge). The pellet of cells was discarded. The supernatant contained the channel-forming activity. It was mixed in a ratio of 1 part supernatant to 9 parts ether and kept overnight at 20 °C. The precipitated protein was dissolved in a solution containing 0·4 % LDAO (N,N-dimethyldodecylamine-N-oxide) and 10 mM Tris/HCl (pH 8), and inspected for channel-forming activity. The protein was subjected to fast protein liquid chromatography (FPLC) across a HiTrap-Q column (Amersham Pharmacia Biotech). The column was washed first with a buffer containing 0·4 % LDAO and 10 mM Tris/HCl (pH 8), and then the protein was eluted with 0·4 % LDAO in 10 mM Tris/HCl (pH 8) using a linear gradient between 0 and 1 M NaCl.
SDS-PAGE.
SDS-PAGE was performed with Tricine-containing gels (Schägger & von Jagow, 1987). The gels were stained with colloidal Coomassie brilliant blue (Neuhoff et al., 1988
) or silver (Blum et al., 1987
). Before separation, the samples were all incubated for 5 min at 100 °C with loading buffer (exempt preparative SDS-PAGE). Preparative SDS-PAGE was used for identification and purification of the channel-forming activity from the organic solvent extracts of whole C. efficiens cells.
Peptide sequencing.
The precipitated protein pellet resulting from the active FPLC fractions or preparative SDS-PAGE was dissolved in 100 µl 70 % (v/v) formic acid containing 10 % (w/v) CNBr (Merck) and incubated in the dark at room temperature for 14 h (Gross, 1967). After lyophilization, the CNBr peptides were dissolved in 20 % (v/v) formic acid and separated by reversed-phase-HPLC (SYCAM), using a Luna C-18 column, 150x1 mm, with a flow rate of 40 µl min1 and a 120 min gradient from 100 % A [0·1 % (v/v) trifluoroacetic acid in water] to 80 % B (0·1 % trifluoroacetic acid in acetonitrile). Collected fractions were subjected to amino acid sequence analysis on a 492 protein sequencer (Applied Biosystems) using the conditions recommended by the manufacturer. The major sequences for the precipitated protein pellet of C. callunae were DLSLLADNLDDYSTFGKNIGTAL and IPDLLKGIIAFFENFGDLAETT; the main sequence for the C. efficiens protein was DLSLLKDSLSDFATLGKN.
Lipid bilayer experiments.
The methods used for black lipid bilayer experiments have been described previously (Benz et al., 1978; Benz, 2003
). The experimental set-up consisted of a Teflon cell with two water-filled compartments connected by a small circular hole. The hole had an area of about 0·4 mm2. Membranes were formed across the hole using a 1 % solution of diphytanoyl phosphatidylcholine (PC; Avanti Polar Lipids) dissolved in n-decane. The temperature was maintained at 20 °C during all experiments. All salts were obtained from Merck (analytical grade). They were used unbuffered. The electrical measurements were performed using Ag/AgCl electrodes (with salt bridges) connected in series to a voltage source and a home-made current-to-voltage converter made with a Burr Brown operational amplifier. The amplified signal was monitored on a storage oscilloscope (Tektronix 7633) and recorded on a strip chart or tape recorder.
The zero-current membrane potentials were measured as described previously (Benz et al., 1979). The membranes were formed in a 100 mM KCl solution containing a predetermined protein concentration so that the membrane conductance increased about 100- to 1000-fold within 1020 min after membrane formation. At this time the instrumentation was switched to the measurements of the zero-current potentials and the salt concentration on the cis side of the membrane was raised by adding small amounts of concentrated salt solutions. The polarity was connected to the trans side. The zero-current membrane potential reached its final value between 2 and 5 min.
Effect of negatively charged groups attached to the channel mouth.
Negative charges at the pore mouth result in substantial ionic-strength-dependent surface potentials at the pore mouth that attract cations and repel anions. Accordingly, they influence both single-channel conductance and zero-current membrane potential. A quantitative description of the effect of charges on the single-channel conductance may be given by the following considerations. The first one is based on the DebeyeHückel theory describing the effect of charges in an aqueous environment. The second treatment was proposed by Nelson & McQuarrie (1975) and describes the effect of charges on the surface of a membrane and does not consider charges attached to a channel. However, this does not represent a serious restriction of its use and we assume here that the charges are localized at the PorH channel. In case of a negative charge, q, in an aqueous environment, a potential
is created that is dependent on the distance, r, from the charge:
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Similarly, the anion concentration , near the charge decreases according to:
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In the following situation we assume that the negative charge is attached to the channel. In such a case its conductance is limited by the accumulated positively charged ions and not by their bulk aqueous concentration. The cation concentration (equation 3) at the mouth of the pore can now be used for calculation of the effective conductanceconcentration curve:
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RESULTS |
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Voltage dependence
In single-channel recordings, channels formed by PorHC.call and PorHC.eff exhibited some flickering at higher voltages, i.e. they showed rapid transitions between open and closed configurations. The voltage-dependent closure of the channels was studied in detail in multi-channel experiments. The channel-forming protein was added at a concentration of 500 ng ml1 to one side of a black PC/n-decane membrane (to the cis side). After 30 min, about 50 channels were reconstituted into the membrane. At that time different potentials were applied to the cis side of the membrane: first 60 mV and then 60 mV. These experiments were repeated with 70, 80 and 90 mV. For both positive and negative potentials applied to the cis side of the membrane the current decreased in an exponential fashion. This result indicated a symmetric response of PorHC.eff to the voltage applied to the membranes. Similar experiments were performed with PorHC.call and a symmetrical response to the applied voltage was also observed (data not shown).
The voltage-dependence experiment, and similar ones, were analysed in the following way. The membrane conductance (G) as a function of voltage, Vm, was measured when the 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, a linear function of the voltage) obtained immediately after the onset of the voltage. The data in Fig. 3(a) (closed circles and squares, for PorHC.eff and PorHC.call, respectively) correspond to the symmetric voltage-dependence of the two cell-wall channels (mean of four membranes) when the proteins were exclusively added to the cis side. The results suggest that PorHC.eff exhibited a somewhat higher voltage-dependence than PorHC.call. The voltage-dependence of the data of Fig. 3(a)
was analysed assuming a Boltzmann distribution between the number of open and closed channels, No and Nc, respectively (Ludwig et al., 1986
). This analysis allowed the calculation of the number of gating charges, n (number of charges involved in the gating process), and the midpoint potential, Vo (potential at which the number of open and closed channels is identical), from a semi-logarithmic plot of the ratio No/Nc, which is given by:
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DISCUSSION |
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Effects of negative charges on the channel properties of PorHC.call
The channels formed by PorHC.call and by PorHC.eff differ somewhat in their ionic selectivity. PorHC.eff forms slightly anion-selective channels despite the fact that the protein is overall acidic (6 negative charges compared to 2 positive ones). Thus it seems that the lysine in position 6 of the mature protein plays a crucial role in the selectivity of the channel because it is absent in the primary sequence of the highly homologous PorHC.call. The data in Table 1 demonstrate that the single-channel conductance of the channels formed by PorHC.call is not a linear function of the bulk aqueous concentration. Instead, we observed a dependence of the single-channel conductance on the square root of the salt concentration in the aqueous phase. This means, (i) that the cation specificity of PorHC.call is not related to the presence of a binding site because saturation would be expected, and (ii) that negative charges are involved in ion selectivity as we and others have demonstrated previously for a variety of membrane channels (Menestrina & Antolini, 1981
; Benz et al., 1989
; Benz, 1994
), which also includes mycobacterial porins (Trias & Benz, 1993
, 1994
; Lichtinger et al., 1998
). When we apply equations (1) to (5) to the conductance of PorHC.call we receive a reasonable fit of the data in Table 1
if the channel has a diameter of about 2·2 nm and 1·6 negative charges (q=2·4x1019 As) are attached to the channel mouth. The results of this fit are shown in Fig. 5
. The solid line represents the fit of the single-channel conductance versus concentration by using the Nelson & McQuarrie (1975)
treatment and the parameters mentioned above together with a single-channel conductance, G0, of 2·8 nS at 1 M KCl. The broken line corresponds to the single-channel conductance of PorHC.call without charges, i.e. it shows a linear relationship between the aqueous salt concentration and single-channel conductance. It is noteworthy that the properties of PorAC.glut from C. glutamicum are also controlled by charges (2 negative charges; q=3·2x1019 As) using the same treatment. Interestingly, the diameter of channels formed by PorAC.glut is very similar to those of PorHC.call. The precise number of charges involved in the charge effect is somewhat difficult to determine because of the use of either NelsonMcQuarrie or DebeyeHückel methods, which results in a difference in the potential by a factor of two. In one case the charge is in an aqueous environment (i.e. the charge is q) and in the other the charge on the surface of the membrane creates an image charge on the other side (i.e. the charge is 2q). The decay of the potential in the aqueous phase is, on the other hand, independent from the localization of the charge, which means that the diameter of the channels is precise.
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ACKNOWLEDGEMENTS |
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Received 20 January 2005;
revised 21 March 2005;
accepted 1 April 2005.
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