Two small polypeptides, PorA and PorH, are known to form cell wall channels in Corynebacterium glutamicum and in Corynebacterium efficiens. The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels of Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae need the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome of Corynebacterium callunae suggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using a porH-and porAdeficient C. glutamicum strain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complemented porH-and porA-deficient C. glutamicum strain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins of C. glutamicum revealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channelforming components identified in C. glutamicum by Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the ؊35 and ؊10 regions of the promoter. The results demonstrate that the cell wall channels within the genus Corynebacterium may be formed by two-component oligomers.

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.