Search

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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).

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.

Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective channels, which had an average single-channel conductance of 3.0 nS in 1 M KCl. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated negative point charge effects on the channel properties. The analysis of the concentration dependence of the single-channel conductance using the effect of negative charges on channel conductance suggested that the diameter of the cell wall channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The cell wall channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the conductance of the cell wall channel.

A channel-forming protein was identified in cell wall extracts of the Gram-positive, strictly aerobic bacterium Nocardia farcinica. The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine-containing SDS-PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 3.0 nS in 1M KCl, 10mM Tris-HCl, pH8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single-channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4-1.6nm. Channel-forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water-filled channels that contain negative point charges.