Molecular characterisation of the pifC gene encoding translation initiation factor 3, which is required for normal photosynthetic complex formation in Rhodobacter sphaeroides NCIB 8253

Abstract

In order to determine whether translation initiation events play a selective role in regulating the expression of photosynthetic complexes in the photosynthetic bacterium Rhodobacter sphaeroides, we have undertaken an initial study to investigate the potential role of translation initiation factor IF3, which also behaves as a pleiotropic regulatory factor in some bacteria. Following the isolation and purification of a 24-kDa IF3-like protein (PifC) from R. sphaeroides, we used nested PCR to clone and characterise the encoding gene, pifC (photosynthesis-affecting initiation factor). The 545-bp pifC encodes a protein exhibiting 60% identity (78.6% similarity) with the Escherichia coli IF3 (InfC) protein and, in common with all other IF3 genes identified to date, pifC possesses a rare initiation codon (AUA). Furthermore, in common with IF3, PifC was shown here to perform a discriminatory function towards CUG start codons, confirming its role and function as an IF3 in R. sphaeroides. Insertion of a kanamycin resistance cassette into the 5' end of pifC resulted in a viable phenotype which exhibits growth rates similar to wild type but which possesses reduced bacteriochlorophyll and photosynthetic complexes in semi-aerobic cultures. It is shown here that the mutant is still able to produce a PifC protein but that it possesses reduced IF3 activity. This may account for the viable nature of the mutant strain, and may indicate that the effect of the mutation on photosynthesis can be more severe than shown in the present study. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.