The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum - Immunological localization, cloning and sequencing of its gene porA

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium gluta micum is the subunit of the cell wall channel. Antibodies raised against Po rA specifically detected the protein on the cell surface. PorA was sequence d using Edman degradation and a gas phase sequencer. The primary sequence w as used to create degenerate oligonucleotide primers. The gene of the chann el-forming protein and its flanking regions were obtained by PCR followed b y inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-l ong acidic polypeptide with an excess of four negatively charged amino acid s in agreement with the high cation selectivity of the PorA cell wall chann el. 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 s mallest subunit of a membrane channel known so far and for the first cell w all 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.