Corynebacterium diphtheriae : Identification and Characterization of a Channel-Forming Protein in the Cell Wall

Abstract

The cell wall fraction of the gram-positive, nontoxic Corynebacterium diphtheriae strain C8 r (؊) Tox ؊ ‫؍(‬ ATCC 11913) contained a channel-forming protein, as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent-treated cell walls and in extracts of whole cells obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.

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

“…Participation of fragmentencoded polypeptides complementing the channel deficiency of the C. glutamicum ⌬porH ⌬porA mutant was studied by site-directed mutagenesis. Therefore, the TOPO 2.1 vector with the original PCR amplicon of strain ATCC 11913 (53) was subjected to QuikChange (QC) PCR using Pfu DNA polymerase (Fermentas) and QC primers in Table 2 (e.g., FP Cd_WTQC1/RP Cd_WTQC1) as described by the manufacturer. Amplicons that contained the intended mutations were cut from the TOPO 2.1 plasmid with EcoRI and XbaI and inserted into the backbone of pXMJ19.…”

“…The fragment complements the channel deficiency of the C. glutamicum ⌬porH ⌬porA mutant (53). Gene silencing disclosed that the 59-amino-acid small protein CdPorH, together with CdPorA, accounted for the channel activity.…”

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

“…They show in addition that the PorAPorH channel shares the characteristic fingerprints of channels such as bacterial porin (34) and also mitochondrial porin (35): closure at positive and negative potential with fast and slow kinetics, asymmetric voltage dependence, and multiple conductance states. Similar porin channels can be found in related families of bacteria, C. diphtheria, C. efficiens, and N. farcinia (8,36,37).…”

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

“…In this study, we identified a channel from the cell wall of D. maris DSM 43672. A protein responsible for channel formation was identified with a molecular mass of about 120 kDa, which may be formed from a homooligomer of smaller polypeptides similar to the situation in other members of mycolata, where the cell wall channels are frequently formed by oligomers [18][19][20][21]. According to results of the lipid bilayer conductance measurements, the cell wall channel of D. maris DSM 43672 is wide, water filled and has a high preference for the passage of cations.…”

“…This means that the mycolic acid layer has the same function as the outer membranes of Gram-negative bacteria, which contain porins for the passage of hydrophilic solutes. Accordingly, channel-forming proteins with characteristics similar to the porins of their Gram-negative counterparts have been identified as existing in the cell walls of multiple members of the mycolata, including Mycobacterium chelonae and Mycobacterium bovis [15,16], Nocardia farcinica [17,18], Corynebacterium diphtheriae, Corynebacterium glutamicum, Corynebacterium amycolatum [19][20][21][22][23], Rhodococcus equi and Rhodococcus erythropolis [24,25]. It is therefore likely that channel-forming proteins are widely distributed in the mycolata and are responsible for the uptake of hydrophilic compounds across the mycolic acid layer.…”