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

Abstract

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.

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

“…Ion Channel Activity of in Vivo-and in Vitro-expressed PorA and PorH-In recent years the functionality of PorA and PorH has been studied with protein extract from the cell wall of C. glutamicum, and results indicate the formation of a functional complex out of both proteins (8), in addition to the requirement of mycolic acid modifications (on position Ser-15 in the case of PorA) for pore-forming activity (13).…”

“…In vivo expression and purification of PorA or PorH out of C. glutamicum were carried out as described previously to obtain both proteins with mycolic acid modification (8). Because the in vivo-expressed samples were analyzed in LDAO (0.4%), we kept the same detergent for sample preparation of CF-expressed PorA and T-PorH.…”

“…Recordings were performed in symmetric media containing an unbuffered solution of 1 M KCl (as in Ref. 8). Currents were amplified using an Axon 200B patch-clamp amplifier, filtered at 1 kHz through an a-pole Bessel filter, and digitized at 2 kHz.…”

“…The major porins, PorA (45 amino acids) and PorH (57 amino acids), form cationselective channels only if present together while PorB and PorC form anion-selective channels (8). The x-ray structure of PorB was solved recently, and it was proposed to exist in the membrane as a putative ␣-helical porin (9).…”

Discovery of a cell wall porin in the mycolic‐acid‐containing actinomycete Dietzia marisDSM 43672

“…However, some exhibit cation selectivity (as in D. maris) whereas others are anion-selective, although it is likely that each organism will possess channels with both types of selectivity in order to facilitate uptake of the full range of hydrophilic solutes. More interestingly, some porins appear to work as hetero-oligomers [18,46] whereas others form likely homo-oligomers [47].…”