Mol Gen Genet (1992) 232:7-11 © Springer-Verlag 1992 Clustering of genes involved in nitrate assimilation in the cyanobacterium Synechococcus Ignacio Luque, Antonia Herrero, Enrique Flores, and Francisco Maduefio * Instituto de BioquimicaVegetaly Fotoslntesis,Universidadde Sevillay CS~C,Facultadde Biologia,Apdo. 1113, E-41080-Sevilla, Spain Received August 16, 1991 Summary. A region of the genome of the cyanobacter- ium Synechococcus R2, that bears a cluster of genes in- volved in nitrate assimilation, has been cloned and the relative positions of some of the genes in the region have been determined. Mutations generated by insertion of an antibiotic-resistance gene cassette into the gene encoding nitrite reductase are associated with reduced expression of nitrate reductase; cotranscription of ni- trate assimilation genes in the cluster is inferred from this finding. Key words: Nitrate assimilation genes Synechococcus - Cotranscription - Genetic regulation Introduction Nitrate is the nitrogen source most commonly utilized by cyanobacteria. Nitrate assimilation in these organ- isms takes place via three successive steps: (i) nitrate uptake into the cell by an active transport system, a component of which has been identified as a 48 kDa polypeptide localized to the cytoplasmic membrane (Ma- duefio et al. 1988b; Omata et al. 1989); (ii) nitrate reduc- tion through two reactions that are sequentially cata- lysed by nitrate reductase and nitrite reductase (Flores et al. 1983) and (iii) incorporation of the ammonium resulting from nitrate reduction into carbon skeletons through the glutamine synthetase-glutamate synthase pathway (Meeks et al. 1978; Flores et al. 1983). Synechococcus R2 is a unicellular cyanobacterium that almost exclusively grows on inorganic nitrogen. In Synechococcus, ammonium acts as an effective antago- nist of nitrate utilization, causing repression of the 48 kDa nitrate transport element (Maduefio etal. 1988b), nitrate reductase (Herrero et al. 1981), nitrite * Present address: Universityof Cambridge, Department of Bota- ny, DowningStreet, CambridgeCB2 3EA, UK Offprint requests to: A. Herrero reductase (Herrero and Guerrero 1986) and glutamine synthetase (Vega-Palas et al. 1990). A regulatory gene (ntcA) has recently been identified whose product has a pleiotropic, positive action on the expression of the aforementioned, ammonium-repressible proteins (Vega- Palas et al. 1990). In addition, the gene nrtA encoding the 48 kDa nitrate transport element (Omata 1991), and three genes (narA, narB and narC) involved in the reduc- tion of nitrate to nitrite (Kuhlemeier et al. 1984a, b) have been cloned. Whereas the actual function of narA and narC is presently unknown, narB might correspond to the nitrate reductase structural gene (Andriesse et al. 1990). Finally, an open reading frame has been found upstream of the gene encoding the 48 kDa polypeptide the putative product of which shows homology to nitrite reductase from spinach (Omata 1991). On the other hand, a mutant strain, FM2, derived from Synechococ- cus R2 has been described (Maduefio etal. 1988a), which is unable to assimilate nitrate. It lacks nitrite re- ductase and constitutively expresses nitrate transport and nitrate reductase genes. This report deals with the cloning of the genomic region mutated in strain FM2 and the corresponding wild-type region from Synechococcus R2. We show that the region contains a cluster of genes involved in nitrate assimilation, and present evidence for cotranscription of these genes. Materials and methods The organisms used throughout this work were Synecho- coccus sp. PCC 7942 (also known as Synechococcus R2); Synechococcus A6, a derivative of Synechococcus R2 that bears an Apt-encoding transposon (Tn901) in plasmid pUH24 (van den Hondel et al. 1980); and FM2, a mu- tant strain derived from Synechococcus A6, which as the result of a single mutation exhibits a pleiotropic phe- notype, being unable to use either nitrate or nitrite as nitrogen sources and showing non-detectable nitrite re- ductase activity and constitutive expression of nitrate