The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure.

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA Req (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA Req has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB Req [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.Rhodococcus equi (formerly Corynebacterium equi) is recognized as a significant cause of disease in foals between the ages of 1 and 6 months and is responsible for ca. 3% of global foal mortality (13,33). This organism was first reported as a cause of human disease in 1967 (14), since which time it has emerged as an opportunistic pathogen, notably (but not exclusively) of patients with compromised immunity (25,27,33,52). Moreover, it is likely that the incidence of human infection by R. equi has been underestimated through either misdiagnosis or unwarranted dismissal of the organism as a contaminant diphtheroid (52).In both foals and humans, R. equi disease is typically characterized by bronchopneumonia, although extrapulmonary manifestations are also found (13, 33, 52). R. equi is an intracellular pathogen of alveolar macrophages, and the bacterium is known to enter macrophages via complement receptors following deposition of complement component C3 (25). Once within the macrophage, the bacteria resist the host's killing mechanisms and multiply. However, there is currently little information concerning the specific bacterial factors which facilitate entry into or subsequent survival within macrophages. Persistence within macrophages most likely contributes to the in vivo ...

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.Keywords: cell wall channel; Corynebacterium glutamicum; lipid bilayer membrane; mycolic acid; porin gene.Corynebacterium glutamicum and Corynebacterium callunae are used for the mass production of glutamic acid [1]. They belong, together with other corynebacteria and members of the genera Rhodococcus, Gordona, Tsukamurella, Dietzia, Mycobacterium and Nocardia, to the broad and diverse group of mycolic acid-containing actinomycetes, the mycolata. Besides the thick peptidoglycan layer, the mycolata contain large amounts of lipids in the form of mycolic acids in their cell walls [2±5]. The mycolic acids are linked through ester bonds to the arabinogalactan attached to the murein of the cell wall [6]. The chain length of these 2-branched, 3-hydroxylated fatty acids varies considerably within the mycolic acid-containing taxa. Thus, especially long mycolic acids have been found in mycobacteria (60±90 carbon atoms) and tsukamurella (64±74 carbon atoms); they are medium-sized in gordona (52±66 carbon atoms) and nocardia (46±58 carbon atoms), and small in corynebacteria (22±38 carbon atoms) [6±11]. The permeability of the cell wall of Mycobacterium chelonae is unusually low [12], presumably because the mycolic acid layer represents a second permeability barrier besides the cytoplasmic membrane similar to the outer membrane of Gram-negative bacteria [13,14].If the mycolic acid layer represents a permeability barrier on the surface of the mycolata, water-filled channels will be needed to allow the permeation of hydrophilic solutes. In agreement with this, channels have been identified in the cell wall of M. chelonae [15,16], Mycobacterium smegmatis [17], Nocardia farcinica [18], Mycobacterium tuberculosis [19±21] and C. glutamicum [22,23]. These channels are wide and water-filled and they contain negative point charges, which results in cation selectivity. Our observations could imply that cell wall porins may be present in all...

The gram-positive bacterium Mycobacterium phlei was treated with detergents. Reconstitution experiments using lipid bilayers suggested that the detergent extracts contain a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with an apparent molecular mass of about 135 kDa. The channel-forming unit dissociated into subunits with a molecular mass of about 22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel conductance of 4.5 nS in 1 m KCl and is highly voltage-dependent in an asymmetric fashion when the protein is added to only one side of the membrane. Zero-current membrane potential measurements with different salts implied that the channel is highly cation-selective because of negative point charges in or near the channel mouth. Analysis of the single-channel conductance as a function of the hydrated cation radii using the Renkin correction factor and the effect of the negative point charges on the single-channel conductance suggest that the diameter of the cell wall channel is about 1.8 to 2.0 nm. The channel properties were compared with those of other members of the mycolata and suggest that these channels share common features. Southern blots demonstrated that the chromosome of M. phlei and other mycolata tested contain homologous sequences to mspA (gene of the cell wall porin of Mycobacterium smegmatis).