MOLECULAR CHARACTERIZATION OF THE PIFC GENE ENCODING TRANSLATION INITIATION-FACTOR-3, WHICH IS REQUIRED FOR NORMAL PHOTOSYNTHETIC COMPLEX-FORMATION IN RHODOBACTER-SPHAEROIDES NCIB-8253

In order to determine whether translation initiation events play a sel ective role in regulating the expression of photosynthetic complexes i n the photosynthetic bacterium Rhodobacter sphaeroides, we have undert aken 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 exhibi ting 60% identity (78.6% similarity with the Escherichia coil IF3 (Inf C) protein and, ill common with all other IF3 genes identified to elat e, pifC possesses a rare initiation codon (AUA). Furthermore. in commo n with IF3, PifC was shown here to perform a discriminatory function t owards 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 r ates similar to wild type but which possesses reduced bacteriochloroph yll rind photosynthetic complexes in semi-aerobic cultures. It is show n here that the mutant is still able to produce a PifC protein but tha t it possesses reduced IF3 activity. This may account for the viable n ature of the mutant strain, and may indicate that the effect of the mu tation on photosynthesis can be mon severe than shown in the present s tudy. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.