Mol Gen Genet (1989) 215:345-348 © Springer-Verlag 1989 Short communications Identification of two fix loci controlling the expression of nif genes in Rhizobium meliloti 41 Z. Banfalvi, V. Petkova, M. Lados, K. Slaska-Kiss, P. Putnoky, C.H. Ung, and A. Kondorosi* Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, PO Box 521, H-6701, Hungary Summary. Recently, Fix- mutants of Rhizobium meliloti 41 defective for nifHDK transcription in the bacteroid state have been described. Two of these mutants have been used to identify bacterial genes involved in the regulation of n/f gene expression. A nifA:: lacZ fusion was introduced into the mutant strains and/~-galactosidase activity was assayed in nodule bacteria, as well as in bacteria grown under micro- aerobic conditions. One of the mutants did not express the nifA gene in symbiosis, suggesting that the gene inactivated by mutation fix-24 is involved in controlling the expression of the nif structural genes via the regulatory gene nifA. The mutation fix-24 also impaired the expression of nifA under microaerobic conditions. These data are in agreement with earlier findings that low oxygen concentration may serve as a signal for n/f gene expression in symbiosis. The fix gene marked by the mutation fix-24 might be a positive regulator of nifA expression in R. meliloti 41. The other mutation (fix-25) represented another cluster off ix genes which also affected the expression of nifA. This influence, however, was specific for symbiosis. The fix genes OCix-24, fix-25) were localized on the symbiotic megaplasmid pRme41b. The two genes are 10 kb apart from each other and are located at 200 kb downstream of the n/f structural genes in R. meliloti 41. Key words: Rhizobium- Symbiosis- Oxygen regulation - n/f genes - Fix mutants In nitrogen fixing microorganisms the conversion of molec- ular nitrogen into ammonia is catalyzed by the nitrogenase enzyme complex. The synthesis and functioning of nitrogen- ase is determined by the nitrogen fixation (n/f) genes which are expressed only under certain conditions. In Rhizobium meliloti, the symbiotic partner of Medi- eago sativa (alfalfa), effective nitrogen fixation takes place only in root nodules, special plant organs developed as a result of the host specific Rhizobium-plant interaction. The mfgenes of R. meliloti, together with the fix genesfixABCX and the nodulation genes (host specific - hsn and "com- mon" nod) are located on one of the two megaplasmids characteristic for R. meliloti (Banfalvi et al. 1981, 1985; Ro- senberg et al. 1981 ; Kondorosi et al. 1984; Earl et al. 1987; Dusha et al. 1987). Although R. meliloti does not fix nitro- Offprint requests to .' A. Kondorosi gen ex planta, the mf structural genes (nifHDK) can be activated by the NtrC protein when the cells are grown in nitrogen limiting media. For symbiotic nitrogen fixation, however, the NtrC protein is not required (Szeto et al. 1987). The n/f genes of R. meliloti are activated also by low oxygen concentration. Under these conditions, the ex- pression of nifA is independent of ntrC and the transcrip- tion of nifA starts at the nifA promoter (Ditta et al. 1987). This promoter is active in symbiosis as well (Kim et al. 1986; Buikema et al. 1985). Recently, in our laboratory (Putnoky et al. 1988) nod- ules induced by 64 random Fix- mutants of R. meliloti 41 were assayed for the ability to express 'late' nodulin genes of M. sativa (leghemoglobin and nodulin-25 genes) and two of the structural nitrogenase genes (nifHD) of R. meliloti. Based on Northern hybridizations using the Lb, nod-25 and nifHD gene probes, six Fix- mutants defective for nifHD transcription in bacteroid form have been identi- fied. Two of them (AK1487, AKI490) were obtained by random Tn5 mutagenesis (Kondorosi et al. 1984). These mutants have now been used to identify bacterial genes involved in n/f regulation. A preliminary account of this work was presented at the 7th International Congress on N2 Fixation (Kondorosi et al. 1988). Using different ap- proaches, very recently two other groups have also identi- fied common regulatory elements controlling symbiotic and microaerobic induction of nifA in R. meliloti 102F34 and 2011 (Virts et al. 1988; Kahn et al. 1988). The fix genes mutated in AK1487 (fix-24) and AK1490 (fix- 25) control the expression ofnif structural genes via nifA The lack of nifHD expression in the mutant bacteroids was originally detected by Northern hybridization. To gain fur- ther support for the inability of mutants AK1487 and AK1490 to express ntf genes, plasmid pRKP9 carrying the nifH: :lacZ fusion (Szeto et al. 1987) was introduced into the two mutants and the/%galactosidase activities of the nodules induced by the transconjugants were assayed. No appreciable nifH expression was detected by this method in AK1487. The fl-galactosidase activity of the AK1490 nodules was 20% of that found in the wild-type bacteroids. The reduced level of nifH expression, however, was specific for the mutation fix-25 (AK1490). Under the same condi- tions, 2 weeks after inoculation no alteration could be de- tected in a nifD:: Tn5 mutant (ZB277; Banfalvi et al. 1983) used as a control in mfexpression experiments (Table 1).