Gene expression and control of enzymes for synthesis of magnesium protoporphyrin monomethyl ester in Rhodobacter sphaeroides.

The photosynthetic gene cluster of R sphaeroides is encoded on a 45kb region of the genome and is represented by 8 overlapping inserts cloned in the mobilisable vector pSUP202 [ l ] . Characterisation of the cluster by a variety of techniques, including complementation analyses and study of accumulated products of bacteriochlorophyll synthesis in mutants, provides a physical map (Figure 1) and allotment of 9-11 genes on the bacteriochlorophyll pathway (Figure 2). The magnesium insertion step is regulated by light and oxygen at the genetic and enzyme level, but has not been characterised further because magnesium chelatase from these microorganisms cannot be assayed in a cell-fiee system. Magnesium chelatase activity has previously been demonstrated in whole cells of wild-type R sphaeroides supplied with exogenous protoporphyrin but the product obtained was magnesium protoporphyrin monomethyl ester and not magnesium rotoporphyrin [3]. With magnesium protoporphyrin as substrate, lowever, magnesium protoporphyrin monomethyl ester formation was readily demonstrated, both in whole cells, and in cell-free extracts [4]. This suggested that the magnesium insertion and methylation steps were closely linked either as a multienzyme complex or as closely coupled sequential reactions. Further studies are now described with mutants obtained by Tn5 mutagenesis [l] which inactivates the named gene. These are designed to advance our understanding of the function and interrelationships of the diffent genes identified at the protoporphyrin + + magnesium protoporphyrin monomethyl ester steps (Figure 2). HUNTER*