Identification and characterization of symbiotic genes on the Rhizobium leguminosarum pre sym-plasmid

Bacteria of the genera Rhizobium and Bradyrhizobium are unique in their quality to form nitrogen-fixing root nodules in symbiosis with leguminous plants. In fast-growing Rhizobium bacteria the genes involved in host recognition and nodule development ( nod ) and in nitrogen fixation ( nif or fix ) are located on large sym -plasmids (for recent review see e.g. Ausubel, 1984).The aim of the present investigations was to identify symbiotic genes in R.leguminosarum PRE and to study their expression in bacteroids. Sym -plasmid clone banks were constructed in order to analyse Its physical organization and to isolate relevant fragments (see chapters II and V). The selection of sym -plasmid regions essential for symbiotic nitrogen fixation was primarily based on DNA sequence homology with specific cloned genes (probes) from other Rhizobium species or Klebsiella .In chapter II the detection of the nitrogenase structural genes by Southern hybridization with a R.melilotinif HDK probe, pRmR2 (Ruvkun and Ausubel, 1980) is described. The nif HDK operon was functionally characterized by site-directed transposon Tn5 mutagenesis and by mapping of protein coding regions after expression of cloned Rhizobium DNA sequences in E.coli mini cells.The nif -regulatory nif A gene, which in K.pneumoniae is involved in the activation of all other nif operons, also appears to be evolutionary conserved among nitrogen-fixing organisms (Dixon, 1984; Ausubel etal ., 1985). In chapters III and IV the identification and characterization of the R.leguminosarum PRE nif A locus is reported. Significant homology was found with K.pneumoniaenif A (Riedel etal ., 1979), a R.meliloti symbiotic regulatory gene and E.colintr C, a gene involved in the regulation of several nitrogen assimilation pathways (Szeto etal ., 1984). Tn5 insertions in the nif A gene resulted in mutants which produced ineffective root nodules with bacteroids in which the synthesis of components I and II of nitrogenase encoded by the nif HDK operon was inhibited. This observation confirmed that the nif A type locus in the sym plasmid of R.leguminosarum has indeed a function in the regulation of the expression of the nitrogenase proteins. The regulatory nif A gene in PRE encodes a 59 kD protein. There was no functional complementation of Rhizobiumnif A mutations by the K.pneumoniaenif A polypeptide in bacteroids or in free living cultures. In addition a coding region immediately downstream from nif A hybridized specifically with R.leguminosarum 248 fix Z, a homologue of the K. pneumoniaenif B locus involved in FeMo-cofactor production (Rossen etal ., 1984).Furthermore, as described in chapter V, a 10 kb nod region has been mapped on the PRE sym -plasmid approximately 7 kb downstream from the nif B homologous gene, based on the homology with an R.meliloti probe carrying nod-genes pRmSL26 (Long etal ., 1982). Three different classes of Nod phenotypes, i.e. deficient, reduced and delayed in nodulation, resulted from Tn5 mutations in this region, and confirmed that this region of the sym plasmid contains nodulation genes.Besides the determination of interspecies DNA homologies, we also used an alternative approach for the detection of sym genes. This was based on the observation of Krol (1982) that certain sym -plasmid regions are exclusively transcribed during endosymblosis. In chapters Ill and V we show that screening of a sym -plasmid clone bank of PRE with labeled bacteroid RNA from 17 days old pea root nodules resulted in the isolation of two non overlapping stretches of DNA each about 50 kb long and including the nif HDK operon and the nif A - nif B - nod cluster respectively.Subfragments of the two sym -plasmid segments showed different levels of hybridization with bacteroid RNA suggesting the presence of a number of different genes. Tn5 mutations in these subfragments further demonstrated that indeed they carry different functional fix genes.A contiguous cluster of such fix loci has been analysed in more detail and was shown to encode 23, 29, 42 and 50 kD polypeptides (chapter IV). This region of the PRE sym -plasmid resembles the fix ABC operon in R.meliloti , as judged from hybridization data, molecular weights of proteins and the location upstream from nif A - nif B (Ausubel etal ., 1985; Weber etal ., 1985); the 23 kD fix gene presumably represents a distinct transcriptional unit.Fig. 1 In chapter V gives an overall picture of the present knowledge of the organization of the symbiotic genes on the sym -plasmid of R.leguminosarum PRE. The genetic organization of sym -plasmids has been compared for different rhizobial species; remarkable similarities exist e.g. conservation of the fix ABC - nif A - nif B cluster, but in general the R.leguminosarum PRE sym -plasmid shows larger distances between and shifts in the location of symbiotic genes.