Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the Genus Corynebacterium

摘要

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 porA-deficient 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 channel-forming 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.