Molecular characterization and organization of porin from Rhodobacter capsulatus strain 37B4.

The primary and atomic structures of the porin protein from Rhodobacter (Rb.) capsulatus strain 37b4 were determined several years ago by peptide sequencing and X-ray crystallography. In this work the gene encoding this porin (named porCa) was cloned and sequenced. The porin open reading frame encodes 320 amino acids-a mature protein of 300 residues (molecular mass 31 552 kDa) and a presequence of 20 amino acids. Our deduced amino-acid sequence was directly confirmed by purifying the porin protein from the same bacterial strain and sequencing the amino terminus as well as several peptides derived from trypsin digestion. However, comparison of this deduced amino-acid sequence with the published primary structure of this porin, nominally from the same strain (but cultivated for ca. 30 years in a different laboratory) reveals seven differences in the amino-acid sequence at the following positions in the mature protein (published/present): 59 (Gly/Ala), 123 (Tyr/Asn), 135 Ser/Thr), 189 (Ile/Val), 196 (Asn/His), 231 (Ala/Thr) and 238 (Ser/deleted). Surprisingly, analysis of the positioning of these mutations revealed that they are located exclusively on transmembrane strands, with two of them deeply buried within the structure. These mutations may in fact have only marginal influence on porin structure and function. Northern blot analysis revealed that porCa encodes an RNA transcript of 1070 nucleotides. No differential response in the abundance or size of this mRNA was seen upon growth under phototrophic/anaerobic vs. chemotrophic/aerobic conditions, under high or low osmotic pressure. Primer extension experiments revealed a transcription start site 73 bases upstream from the ATG translation start, juxtaposed to the identified putative promoter region. Fusion of lacZ with this putative promoter region (using a 288-bp upstream region) revealed similar promoter activity in beta-galactosidase assays under both physiological conditions tested, again suggesting that this gene is constitutively expressed. The molecular genetic characterization described in this work opens the way for structure-function studies by site-directed mutagenesis.