PorH, a new channel-forming protein present in the cell wall of Corynebacterium efficiens and Corynebacterium callunae

Peter Hünten1, Bettina Schiffler1, Friedrich Lottspeich2 and Roland Benz1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Corynebacterium callunae and Corynebacterium efficiens are close relatives of the glutamate-producing mycolata species Corynebacterium glutamicum. The properties of the pore-forming proteins, extracted by organic solvents, were studied. The cell extracts contained channel-forming proteins that formed ion-permeable channels with a single-channel conductance of about 2 to 3 nS in 1 M KCl in a lipid bilayer assay. The corresponding proteins from both corynebacteria were purified to homogeneity and were named PorHC.call and PorHC.eff. Electrophysiological studies of the channels suggested that they are wide and water-filled. Channels formed by PorHC.call are cation-selective, whereas PorHC.eff forms slightly anion-selective channels. Both proteins were partially sequenced. A multiple sequence alignment search within the known chromosome of C. efficiens demonstrated that it contains a gene that fits the partial amino acid sequence of PorHC.eff. PorHC.call shows high homology to PorHC.eff. PorHC.eff is encoded in the bacterial chromosome by a gene that is localized within the vicinity of the porA gene of C. efficiens. PorHC.eff has no signal sequence at the N terminus, which means that it is not exported by the Sec-secretion pathway. The structure of PorH in the cell wall of the corynebacteria is discussed.


Abbreviations: LDAO, N,N-dimethyldodecylamine-N-oxide; PC, diphytanoyl phosphatidylcholine

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ871585 and AJ871586.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In 2002, three glutamic-acid-producing coryneform strains, belonging to the genus Corynebacterium, were isolated from soil and vegetable samples. Phylogenetic studies, based on 16S rRNA analysis, demonstrated that the nearest relatives of these strains were Corynebacterium glutamicum and Corynebacterium callunae, which are known as glutamic-acid-producing species. The most significant characteristics of the newly found strains, named Corynebacterium efficiens, was the production of acid from dextrin (Fudou et al., 2002). C. glutamicum is used for industrial production of L-glutamate, L-lysine and other amino acids through fermentation processes (Udaka, 1960; Gutmann et al., 1992; Keilhauer et al., 1993; Sahm et al., 1996; Eggeling & Sahm, 1999). The range of temperature for the growth of most corynebacteria is between 30 and 37 °C, with the exception of C. efficiens (Fudou et al., 2002) which can grow up to 45 °C. This feature is beneficial from an economic point of view; the need for a cooling system in industrial fermenters is reduced using this bacterium, which means that the costs for the production of L-glutamic acid could be reduced.

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 22–38 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
C. efficiens AJ 12310 (obtained from DSMZ, Braunschweig, Germany) was routinely grown in BHI medium (Difco) and C. callunae ATCC 15991 (obtained from DSMZ) was grown in double yeast tryptone (2x YT; BIO 101) medium, both at 30 °C.

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 5–8 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 min–1 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 10–20 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 Debeye–Hückel 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 {Phi} is created that is dependent on the distance, r, from the charge:

{mic1512429E001}
where {varepsilon}0 (=8·85x10–12 F m–1) and {varepsilon} (=80) are the absolute dielectric constant of vacuum and the relative constant of water, respectively, and lD is the so-called Debeye length that controls the decay of the positive potential (and that of the accumulated positively charged ions) in the aqueous phase:

{mic1512429E002}
where c is the bulk aqueous salt concentration, R is 8·3 J mol–1 K–1, T is 293 K and F is 96485·3 As mol–1 (RT F–1=25·2 mV at 20 °C). The potential {Phi} created by a negative charge on the surface of a membrane is twice that of equation (1) caused by the generation of an image force on the opposite side of the membrane (Nelson & McQuarrie, 1975; Benz et al., 1994). The concentration of the monovalent cations near the charge increases because of the negative potential. Their concentration is in both cases (Debeye–Hückel or Nelson–McQuarrie) dependent on the potential {Phi} and given by:

{mic1512429E003}

Similarly, the anion concentration {mic1512429E008}, near the charge decreases according to:

{mic1512429E004}

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 conductance–concentration curve:

{mic1512429E005}
where G0 is the concentration-independent conductance of the channel.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification of PorHC.call and PorHC.eff
Proteins within the organic solvent extract of whole C. callunae and C. efficiens cells were precipitated with ether in the cold. The precipitate was dissolved in the detergent LDAO and inspected for channel-forming activity using the lipid bilayer assay. The detergent solution had a high channel-forming activity, and channels with a conductance between 2 and 6 nS in 1 M KCl were formed under these conditions. Purification of the channel-forming proteins from C. callunae and C. efficiens was performed by FPLC across a HiTrap Q column. Fig. 1(a), lane 2 shows the protein composition of the organic solvent extract of C. callunae that was applied to the column. The column was first washed with buffer and then eluted with buffer supplemented with increasing concentration of NaCl. Pure 6 kDa protein was eluted at an NaCl concentration of 0·23 M. The 6 kDa protein of C. efficiens was not completely pure after FPLC across a HiTrap Q column. Final purification was achieved by preparative SDS-PAGE of the precipitated organic solvent extract that contained the channel-forming activity (see Fig. 1b).



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Fig. 1. (a) Tricine (10 %) SDS-PAGE of the C. callunae PorH purification procedure. The gel was stained with colloidal Coomassie. Lanes: 1, low molecular mass marker; 2, 15 µl ether-precipitated extract dissolved in 0·4 % LDAO, treated for 10 min at 100 °C with 5 µl sample buffer; 3, 15 µl fraction 17 from the Hitrap-Q FPLC column, treated for 10 min at 100 °C with 5 µl sample buffer. (b) 12 % Tricine (12 %) SDS-PAGE of C. efficiens PorH obtained by elution of the 6 kDa band from preparative SDS-PAGE. The gel was stained with silver. Lanes: 1, low molecular mass marker; 2, 15 µl ether-precipitated extract dissolved in 0·4 % LDAO, treated for 10 min at 100 °C with 5 µl sample buffer; 3, 3 µg pure 6 kDa protein solubilized for 10 min at 100 °C with 5 µl sample buffer.

 
Single channel analysis of PorHC.call and PorHC.eff
Fig. 2(a) shows a single-channel recording of a PC membrane in the presence of the pure 6 kDa protein of C. callunae, which was added to a black membrane in a concentration of about 10 ng ml–1. The single-channel recording demonstrates that PorHC.call formed defined channels. The single-channel conductance of most channels was about 3 nS in 1 M KCl. Only a minor fraction of channels with different conductance values was observed (see Fig. 2a). It is noteworthy that the channels formed by PorHC.call of C. callunae had a long lifetime, similar to those that have been detected previously for porins of Gram-negative (Benz, 1994) and Gram-positive bacteria (Lichtinger et al., 1999). All these porins form channels in lipid bilayer membranes with long lifetimes at low transmembrane potential (mean lifetime at least 5 min). However, voltage-dependence closure was observed for PorHC.call of C. callunae for voltages higher than about 30–40 mV (see below). Channels formed by PorHC.eff had a very similar lifetime compared to those formed by PorHC.call, as the single-channel recording of Fig. 2(b) clearly indicates. The only exception was the occurrence of two maxima in the histogram of the single-channel distribution (see Fig. 2b). These two maxima (2·3 and 4·7 nS in 1 M KCl) most probably reflect the reconstitution of two channels at once, because the conductance of the right-side maximum was twice of that of the left side.



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Fig. 2. Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of PC/n-decane in the presence of the pure cell-wall proteins of (a) C. callunae and (b) C. efficiens. P(G) is the probability that a given conductance increment G is observed in the single-channel experiments. It was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. The aqueous phase contained 1 M KCl and 10 ng cell-wall proteins ml–1. The applied membrane potential was 20 mV at 20 °C. The mean single-channel conductance was 3·0±0·4 nS for 139 single-channel events in PorHC.call and 2·3±0·3 nS for 126 single-channel events (left-hand side maximum in Fig. 2b) in PorHC.eff. The insets display the single-channel recording of PC/n-decane membranes in the presence of the pure 6 kDa proteins from C. callunae (a) and C. efficiens (b).

 
Single-channel experiments were also performed with salts other than KCl to obtain some information on the size of the channels formed by PorHC.call and PorHC.eff and their ion selectivity. The results are summarized in Table 1. The conductance sequence of the different salts within the channel formed by PorHC.call was RbCl{approx}KCl>K acetate>NaCl>LiCl>N(CH3)4Cl>N(C2H5)4Cl, which means that the single-channel conductance followed approximately the aqueous mobility of the different cations in the aqueous phase. Presumably, this means that the influence of cations on the conductance of the channel in different salt solutions was more substantial than that of anions (Table 1), suggesting a cation selectivity of the channel. Table 1 also shows the mean single-channel conductance, G, of PorHC.call as a function of the KCl concentration in the aqueous phase. Similarly, as in the case of many porin channels of Gram-positive bacteria (Trias & Benz, 1993, 1994; Riess et al., 1998; Lichtinger et al., 1999), the conductance was not a linear function of the KCl concentration, which is characteristic for the presence of net charges in or near the channel (Trias & Benz, 1994; Lichtinger et al., 1999).


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Table 1. Mean single-channel conductance, G, of PorHC.call and PorHC.eff in different salt solutions

The membranes were formed of PC dissolved in n-decane. The aqueous solutions were unbuffered and had a pH of 6, unless otherwise indicated. The concentration of PorHC.call and PorHC.eff was about 10 ng ml–1. Note that the mean single-channel conductance of PorHC.eff always corresponded to the left-side maximum of the histograms. The applied voltage was 20 mV and the temperature was 20 °C. The mean single-channel conductance, G, was calculated from at least 80 single events. NM, Not measured.

 
Table 1 demonstrates that the single-channel conductance of the channels formed by PorHC.eff was in the same range as that measured for PorHC.call. However, it seems that the ion selectivity of PorHC.eff was somewhat different because the conductance of the channels in LiCl was higher than in K-acetate (Table 1). Furthermore, the conductance of salts containing tetraalkylammonium ions was more or less independent of the size of the cation. These results suggested that PorHC.eff could form anion-selective channels. Zero-current membrane potential measurements were performed in the presence of KCl gradients to check such a possibility. Fivefold KCl gradients (100 versus 500 mM) were established across lipid bilayer membranes which contained about 100–1000 PorHC.eff or PorHC.call channels. The measurements with PorHC.eff resulted in an asymmetry potential of about –6 mV at the more dilute trans side (mean of four measurements). This result indicated some preferential movement of chloride over potassium ions through the PorHC.eff channel at neutral pH. Similar experiments with PorHC.call resulted in an asymmetry potential of about 28 mV at the more dilute side. The zero-current membrane potentials were analysed using the Goldman–Hodgkin–Katz equation (Benz et al., 1979). The ratio of the potassium permeability, PK, divided by the chloride permeability, PCl, was about 0·7 and 7 for PorHC.eff and PorHC.call, respectively, which indicated a small anion selectivity for PorHC.eff and cation selectivity for PorHC.call (see also Discussion).

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 ml–1 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:

{mic1512429E006}
where G in this equation is the conductance at a given membrane potential, Vm, and G0 and Gmin are the conductance at zero voltage and very high potentials, respectively. The open to closed ratio of the channels, No/Nc, is given by:

{mic1512429E007}
where F (Faraday's constant: 96485·3 C mol–1), R (gas constant: 8·3 J mol–1 K–1) and T (absolute temperature: 293 K) are standard symbols, n is the number of gating charges moving through the entire transmembrane potential gradient for channel gating (i.e. a measure for the strength of the interaction between the electric field and the open channel) and V0 is the potential at which 50 % of the total number of channels are in the closed configuration (i.e. No/Nc=1). Semi-logarithmic plots of the data given in Fig. 3(a) using least-squares fits (see Fig. 3b) show that they could be fitted to straight lines. The slope of the lines was such that an e-fold change of No/Nc occurred when the voltage was changed by about 16 mV (PorHC.call) or 14 mV (PorHC.eff). This means that the number of gating charges is approximately n=1·6 in the first case and n=1·8 in the latter case (because RT/F=25·2 mV; see Fig. 3b). The broken numbers could mean that the gating charges do not move through the entire membrane thickness. Whereas the voltage dependence (the slope of the lines of Fig. 3a) was approximately similar for both channels, the midpoint potential V0 (i.e. No/Nc=1) differed somewhat for PorHC.eff (V0{approx}±50 mV) and PorHC.call (V0{approx}±80 mV) (see Fig. 3b).



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Fig. 3. (a) Ratio of the conductance G at a given membrane potential (Vm) divided by the conductance G0 at 10 mV as a function of Vm. {blacksquare}, Measurements in which PorHC.call was added to the cis side of a membrane; {bullet}, measurements in which PorHC.eff was added to the cis side of membranes. The membrane potential always refers to the cis side of the membrane. The aqueous phase contained 1 M KCl and 500 ng porin ml–1. The membranes were formed from PC dissolved in n-decane. T=20 °C. Means of values obtained with four different membranes are shown. (b) Semilogarithmic plot of the ratio No/Nc as a function of Vm. The data were taken from (a). The slope of the straight lines obtained by least squares fits is such that an e-fold change of No/Nc is produced by a change in Vm of about 15–16 mV (PorHC.call) and about 14 mV (PorHC.eff). The midpoint potential of the No/Nc distribution (i.e. No=Nc) is ±80 mV for PorHC.call and ±50 mV for PorHC.eff. For further explanation see text.

 
Partial sequencing of the 6 kDa channel-forming proteins of C. callunae and C. efficiens and identification of porHC.eff within the chromosome of C. efficiens
The 6 kDa channel-forming proteins of C. callunae and C. efficiens were subjected to partial sequencing from the N-terminal end of the mature proteins after CNBr treatment using Edman degradation. Three stretches of 22 and 23 aa (C. callunae) and 18 aa (C. efficiens) were resolved. Multiple sequence alignments were performed with the translated known nucleotide sequence of the complete C. efficiens genome (NCBI reference sequence accession number NC_004369). The NCBI BLAST translation tool (Basic Local Alignment Search Tool; Zhang & Madden, 1997; Altschul et al., 1990) showed that the 18 aa stretch of C. efficiens is part of a 57 aa hypothetical protein of C. efficiens (DDBJ/EMBL/GenBank accession no. AJ871586; see Fig. 4a), which we named PorHC.eff. Interestingly, it exhibits only the inducer methionine at the N-terminal end, but no N-terminal extension, which suggests that translation and assembly of the protein could be very similar to that of PorA of C. glutamicum (Lichtinger et al., 2001). The gene porHC.eff consists of 174 bp and encodes a 57 aa acidic polypeptide (6 aspartic and glutamic acids compared to 2 lysines) without a leader sequence. This means that PorHC.eff is not transported out of the cytoplasmic membrane using the Sec apparatus as many other proteins from Gram-positive bacteria are (Freudl, 1992; Lichtinger et al., 2001). A search within the chromosome of C. efficiens demonstrates that porH and the gene encoding PorA of C. efficiens are localized very close to one another (see Fig. 4b). The genes encoding both proteins are only separated by 77 bp and there is no indication of a transcription terminator between them. Thus it seems very likely that both proteins share a common mode of export to the cell wall in C. efficiens and presumably also in C. glutamicum because the chromosome of the latter also contains the porHC.glut gene that has a high degree of homology to porHC.eff. Comparison of the two amino acid stretches (22 and 23 aa) derived from sequencing of PorHC.call with the sequence of PorHC.eff and PorHC.glut suggests that the proteins are highly homologous (see Fig. 4a). PorHC.call is also an acidic protein (8 aspartic and glutamic acids compared to 2 lysines of the partial sequence). The interesting feature of the channels formed by the two homologous proteins is the observation that one protein forms slightly anion-selective channels (PorHC.eff) and the other forms highly cation-selective channels (PorHC.call). This means presumably that their arrangement in the channel-forming complexes may be responsible for their selectivity (see Discussion).



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Fig. 4. (a) Amino acid sequence of PorHC.eff (DDBJ/EMBL/GenBank accession no. AJ871586) and comparison with the partial amino acid sequences of the N terminus of PorHC.call and PorHC.glut (DDBJ/EMBL/GenBank accession no. AJ871585) after CNBr cleavage. The result of amino acid sequencing after CNBr cleavage of PorHC.eff using Edman degradation of the N terminus is underlined. The charged residues of the proteins (+/–) are specified on the top line. Conserved residues in both homologous proteins are shown in bold type. The sequences within the vertical bars are supposed to fold into {alpha}-helices as shown in Fig. 6. (b) Overview of the porH gene locus and its flanking regions within the C. efficiens genome. Putative transcriptional terminators are shown by stem–loop structures, potential ribosome-binding sites with the sequence AGGAG are shaded and a putative promoter is presented by a triangle. Gene names are specified; CE2561 encodes the putative chaperonin GroEL2; CE2562 and CE 2563 encode hypothetical proteins.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cell walls of C. efficiens and C. callunae contain ion-permeable channels formed by the 6 kDa PorH proteins
In previous studies we identified different cell-wall channels in C. glutamicum (Lichtinger et al., 1998, 2001; Costa-Riu et al., 2003a, b). PorAC.glut forms a highly conductive cation-selective channel. Its deletion resulted in a much higher resistance of this bacterium to neutral or positively charged antibiotics, which indicates a lower permeability of the cell wall in the deletion mutant (Costa-Riu et al., 2003a). Nevertheless, growth of the mutant strain was only slightly impaired, in particular in rich medium. A search for another cell-wall channel revealed the existence of the anion-selective PorBC.glut channel (Costa-Riu et al., 2003a, b). This result indicates that the cell wall of C. glutamicum contains several types of channels, as is the case in the outer membrane of Gram-negative bacteria (Benz, 2001) and in the Gram-positive Rhodococcus equi, which is also a member of the mycolata (Riess et al., 2003). In this study, we inspected the cell walls of C. efficiens and C. callunae, which are closely related to C. glutamicum, for the presence of cell-wall channels using the lipid bilayer technique. In organic solvent extracts of whole cells, channels were observed for both organisms that had a molecular mass of about 6 kDa, but were not identical to the well studied PorAC.glut channels. The channel-forming proteins of C. efficiens and C. callunae were purified to homogeneity and were named PorHC.eff and PorHC.call, respectively. Partial N-terminal sequencing of the proteins after CNBr cleavage resulted in three amino acid stretches that allowed the identification of the porH gene within the chromosome of C. efficiens. This gene encodes a 57 aa polypeptide without a leader extension, but it starts with the inducer methionine, which could be cleaved either during maturation or by CNBr. PorHC.call is highly homologous to PorHC.eff and the chromosome of C. glutamicum also contains a homologue gene of a similar protein PorHC.glut (see Fig. 4; DDBJ/EMBL/GenBank accession no. AJ871585), which will be described in detail in a future study. The lack of an N-terminal leader extension suggested that PorHC.eff is not transported via the Sec apparatus out of the cell to reach the cell wall. This is the same situation as for PorAC.glut and considering the genes within the flanking regions of porA and porH it seems likely that their gene products share the same mode of translation, export and assembly, which represents a yet unknown secretion mechanism (Lichtinger et al., 2001).

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=–2·4x10–19 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=–3·2x10–19 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 Nelson–McQuarrie or Debeye–Hückel 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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Fig. 5. Single-channel conductance of PorHC.call as a function the KCl concentration in the aqueous phase ({blacksquare}). The solid line represents the fit of the single-channel conductance data with the Nelson & McQuarrie (1975) formula [equations (1)–(5)], assuming the presence of negative point charges (1·6 negative charges; q=–2·85x10–19 As) at the channel mouth and assuming a channel diameter of 2·2 nm. G, mean single-channel conductance. 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.

 
Arrangement of PorHC.call and PorHC.eff in the cell wall
PorHC.call and PorHC.eff have a rather small molecular mass of about 6 kDa, similar to that of PorAC.glut or PorBC.glut. In general, the molecular masses of cell-wall porins are rather small compared to those of Gram-negative bacterial porins, which range between 30 and 60 kDa (Benz, 1994; Riess et al., 1998). This suggests that the cell-wall channels are formed by oligomers. This has been demonstrated for the subunit of the cation-selective channel of M. smegmatis, which has a molecular mass of about 20 kDa (Niederweis et al., 1999; Stahl et al., 2001) and forms an octamer in the cell wall (Faller et al., 2004). The monomers within the 3D structure of the octamer are arranged in a {beta}-sheet structure similar to that of Gram-negative bacterial porins. However, such an arrangement is rather unlikely for the PorHC.call and PorHC.eff oligomers. Secondary structure predictions suggest that a stretch of about 28 aa (see Fig. 4a) of both proteins form amphipathic {alpha}-helices with about eight windings and a length of 4·2 nm in the mycolic acid layer (see Fig. 6). The arrangement is such that all hydrophilic amino acids are localized on one side of the helix and all hydrophobic ones are on the other side. Comparison of the helical wheels from the two organisms indicates that positively and negatively charged amino acids are balanced for PorHC.eff, whereas the monomer of PorHC.call contains an excess of two negatively charged amino acids. It is noteworthy that this agrees well with the selectivity of both channels (see above). Secondary structure predictions for the homologous porin PorHC.glut of C. glutamicum likewise point to amphipathic {alpha}-helices (data not shown).



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Fig. 6. Schematic prediction of the PorHC.eff and PorHC.call secondary structures. Both molecules can form {alpha}-helices with eight windings corresponding to an overall length of 4·2 nm based on secondary structure predictions of the primary sequence shown in Fig. 4(a) between the two vertical bars. Residues of the heptameric repeats are labelled in the sequence as a–g. The hydrophobic residues are located at positions a, e and d, indicating that they may be oriented towards the mycolic acids (indicated by oval rings). The hydrophilic residues are localized at the positions b, f, c and g and may face the channel lumen. Created with the help of Helical Wheel Java Applet (www.site.uottawa.ca/~turcotte/resources/HelixWheel/).

 
This means that the arrangement of these cell-wall channels is different to MspA of M. smegmatis (Faller et al., 2004). It is possible that the arrangement of PorHC.call and PorHC.eff is associated with the thickness of the cell wall and the length of the mycolic acids of different mycolata. Thus, especially long mycolic acids have been found in mycobacteria and tsukamurellae (60–90 carbon atoms); they are medium-sized in gordonae, nocardiae and rhodococci (about 36–66 carbon atoms) and small in corynebacteria (22–38 carbon atoms) (Yano & Saito 1972; Minnikin et al., 1974, 1982; Minnikin 1987, 1991; Daffé et al., 1990; Holt et al., 1994; Ochi, 1995; Brennan & Nikaido 1995; Liu et al., 1995, 1996; Yassin et al., 1997). This means presumably that the cell walls of corynebacteria are much thinner than those of other mycolata, which is in agreement with structural studies (Marienfeld et al., 1997; Puech et al., 2001). Smaller polypeptides arranged as {alpha}-helices are presumably sufficient to span the mycolic acid layer of corynebacteria. However, this may be tentative and further investigation of the cell-wall proteins of actinomycetes may be necessary to understand the structure and function of the cell-wall channels of the mycolata.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Christian Andersen for helpful discussions and help with the secondary structure predictions of the proteins. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Be 865/9-5) and by the Fonds der Chemischen Industrie.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 20 January 2005; revised 21 March 2005; accepted 1 April 2005.



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