Mol Gen Genet (1984) 196:290-300 © Springer-Verlag 1984 Directed transposon Tn5 mutagenesis and complementation in slow-growing, broad host range cowpea Rhizobium Mittur N. Jagadish and Aladar A. Szalay Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853, USA Summary. Site-directed Tn5 mutagenesis by gene replace- ment method, via homologous recombination, was used to identify symbiotically essential regions in the genome of cowpea Rhizobium spp. IRc78. Transposon insertions with- in the nifK hybridizing region or in the regions spanning 10 kb downstream of the nifK have revealed the presence of functional genes required for nitrogen fixation. Six single Tn5 insertions resulted in nod + fix- phenotypes and one in nod ÷ but reduced fix + phenotype. All seven Tn5 inser- tions were stable before, during and after plant passage. However, IRc78 transconjugants containing duplicated n/f copies, (a normal and a Tn5 inserted copy separated by vector sequences) were unstable. In five IRc78::Tn5 strains, the mutant phenotypes were corrected by an ex- trachromosomally stable vector containing wild type mf al- leles. Our experiments suggest that the correction to nod ÷ fix ÷ phenotype is by complementation although correction by recombination cannot be completely excluded. Introduction Biological nitrogen fixation in the Leguminoceae family is a result of an effective symbiosis between the members of the Gram negative bacterium genus Rhizobium and legume host plants. The process of symbiosis is a complex temporal order of events that include recognition of the symbiotic partners, invasion of root-hair by Rhizobium, root hair cur- ling, formation of infection thread, penetration and branch- ing of infection thread, multiplication of bacteria within the infection thread, division of root cortical cells, release of rhizobia from infection thread into the host cells of the developing nodule, differentiation of Rhizobium into bacter- oids and differentiation of host cells into a mature nodule (Vincent 1974, 1980; Verma and Long 1983). Nodule devel- opment is accompanied by synthesis and expression of sev- eral bacteroid specific products, including nitrogenase as the major component (10%-12%, Whiting and Dilworth 1974), as well as synthesis of the host-specific proteins (Le- gocki and Verma 1979, 1980; Verma and Long 1983). The oxygen binding protein leghemoglobin, essential for protec- tion of the oxygen sensitive enzyme nitrogenase is the as- sembled product of heme and globin components that ap- peared to be encoded by Rhizobium and host plant genes respectively (Godfrey and Dilworth 1971; Leong et al. 1982; Verma and Long 1983). Offprint requests to ." A.A. Szalay The identification and characterization of the bacterial genes and the host-plant genes to establish a correlation between the genetic loci and the multiple events that occur during symbiosis has been the subject of several recent stud- ies (Long etal. 1981; Hirsch etal. 1983; Banfalvi etal. 1981; Corbin et al. 1982, 1983; Scott et al. 1982; Downie et al. 1983; Fuller et al. 1983; Verma and Long 1983). The genus Rhizobium includes fast-growers with a mean generation time of 2-4 h and slow-growers with a mean generation time of 6-8 h (Vincent 1977). While the fast- growers are limited in their host-range, the slow-growers are generally promiscuous in their host range for symbiotic nitrogen fixation. Genetic analyses to study the organization and regula- tion of the nitrogen fixation (n/J) genes have been well estab- lished for a free-living nitrogen fixer Klebsiella pneumoniae (MacNeil etal. 1978; Merrick etal. 1980; Beynon et al. 1983) and for many of the fast-growing Rhizobium species (Meade and Signer 1977; Beringer et al. 1978; Kondorosi et al. 1977; Casadesus and Olivares 1979). Transposon Tn5 which confers resistance to kanamycin, neomycin and G418 antibiotics was used as a mutagenic element to identify the symbiotic genes in the fast-growing species of Rhizobium which include R. leguminosarum (Beringer et al. 1978; Bu- chanan-Wollaston et al. 1980; Downie et al. 1983); R. meli- loti (Banfalvi et al. 1981; Meade et al. 1982; Ruvkun et al. 1982; Corbin et al. 1982; Leong et al. 1982) and R. trifoli (Scott et al. 1982). Among the slow-growing species of Rhi- zobium the genetic analysis has been primarily confined to R. japonicum (Maier and Brill 1976; Haugland and Verma 1981; Stacey et al. 1982; Hennecke 1981; Fuhrmann and Hennecke 1982; Hahn and Hennecke 1984; Cantrell et al. 1983) and very little is known about cowpea rhizobia (" cowpea miscellany") (Ludwig and Signer 1977; Kennedy et al. 1981). Cowpea rhizobia nodulate a broad spectrum of tropical legumes (Ahmad et al. 1981) leading to an effec- tive fixation of atmospheric nitrogen and thus they appear to be very versatile in their host recognition capacity. Despite the differences among fast- and slow-growing rhizobia and K. pnuemoniae there appears to be a high con- servation of the DNA sequences (mf H, D, and K) coding for the structural components of the enzyme complex nitro- genase (Ruvkun and Ausubel 1980; Hadley et al, 1983). A previous report from this laboratory (Hadley et al. 1983) described the identification of the DNA regions that are homologous to K. pneumoniae nitrogenase genes in several cowpea Rhizobium species isolated from various geographi-