THE Q-GENE OF RHODOBACTER-SPHAEROIDES - ITS ROLE IN PUF OPERON EXPRESSION AND SPECTRAL COMPLEX ASSEMBLY

The Q gene of the facultative photoheterotroph Rhodobacter sphaeroides , localized immediately upstream of the oxygen- and light-regulated pu f operon, encodes a 77-amino-acid polypeptide. The 5' and 3' ends of t he 561-bp Q transcript were determined. To gain insight into the role of the and gene product, a number of Q mutations were constructed by o ligonucleotide-directed mutagenesis and subsequent substitution of the mutated form of the gene in single copy for the chromosomal copy via homologous recombination. The resulting mutants can grow photosyntheti cally, with the exception of QSTART, in which the initiation codon for the Q protein was altered. Spectral analysis of the intracytoplasmic membranes showed that one of the missense mutants (QdA) was deficient in the formation of detectable B875 light-harvesting complex (LHC), wh ereas deletion of the stem-loop structure (Qloop) failed to form B800- 850 LHC when grown anaerobically either in the dark or under light int ensity of 100 W/m(2). Other missense mutants (QuA and QuB) contained e ither more B800-850 LHC or more B875 LHC, respectively, than the wild type. Although the levels of puf and puc transcripts isolated from QST ART grown anaerobically on succinate-dimethyl sulfoxide in the dark we re comparable to wild-type levels, no B875 spectral complex was detect ed and there was a greater than 90% reduction in the level of the B800 -850 pigment-protein complex. It has also been confirmed that the ulti mate cellular levels of either the B875 or B800-850 spectral complexes can vary over wide limits without any change in the level(s) of compl ex specific transcripts. When the wild-type Q gene was reintroduced in trans into the Q mutations, QSTART was able to grow photosyntheticall y and both B800-850 and B875 spectral complexes were formed in either QdA or Qloop. Finally, we demonstrated that the level of each puf-spec ific mRNA behaves independently of one another as well as independentl y of the level(s) of and gene-specific mRNA. These results are compati ble with the existence of regulatory sequences affecting the puf mRNA level(s) being localized within the Q structural gene. These results s uggest that Q-specific expression is uncoupled from puf-specific trans cription and that the Q protein is not involved in the regulation of t ranscription of the puf operon but is directly involved in the assembl y of both the B875 and B800-850 pigment-protein complexes.