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

Bacteria of the genera <u>Rhizobium</u> and <u>Bradyrhizobium</u> are unique in their quality to form nitrogen-fixing root nodules in symbiosis with leguminous plants. In fast-growing <u>Rhizobium</u> bacteria the genes involved in host recognition and nodule development ( <u>nod</u> ) and in nitrogen fixation ( <u>nif</u> or <u>fix</u> ) are located on large <u>sym</u> -plasmids (for recent review see <u>e.g.</u> Ausubel, 1984).The aim of the present investigations was to identify symbiotic genes in <u>R.leguminosarum</u> PRE and to study their expression in bacteroids. <u>Sym</u> -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 <u>sym</u> -plasmid regions essential for symbiotic nitrogen fixation was primarily based on DNA sequence homology with specific cloned genes (probes) from other <u>Rhizobium</u> species or <u>Klebsiella</u> .In chapter II the detection of the nitrogenase structural genes by Southern hybridization with a <u>R.meliloti</u><u>nif</u> HDK probe, pRmR2 (Ruvkun and Ausubel, 1980) is described. The <u>nif</u> HDK operon was functionally characterized by site-directed transposon Tn5 mutagenesis and by mapping of protein coding regions after expression of cloned <u>Rhizobium</u> DNA sequences in <u>E.coli</u> mini cells.The <u>nif</u> -regulatory <u>nif</u> A gene, which in <u>K.pneumoniae</u> is involved in the activation of all other <u>nif</u> operons, also appears to be evolutionary conserved among nitrogen-fixing organisms (Dixon, 1984; Ausubel <u>et</u><u>al</u> ., 1985). In chapters III and IV the identification and characterization of the <u>R.leguminosarum</u> PRE <u>nif</u> A locus is reported. Significant homology was found with <u>K.pneumoniae</u><u>nif</u> A (Riedel <u>et</u><u>al</u> ., 1979), a <u>R.meliloti</u> symbiotic regulatory gene and <u>E.coli</u><u>ntr</u> C, a gene involved in the regulation of several nitrogen assimilation pathways (Szeto <u>et</u><u>al</u> ., 1984). Tn5 insertions in the <u>nif</u> 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 <u>nif</u> HDK operon was inhibited. This observation confirmed that the <u>nif</u> A type locus in the <u>sym</u> plasmid of <u>R.leguminosarum</u> has indeed a function in the regulation of the expression of the nitrogenase proteins. The regulatory <u>nif</u> A gene in PRE encodes a 59 kD protein. There was no functional complementation of <u>Rhizobium</u><u>nif</u> A mutations by the <u>K.pneumoniae</u><u>nif</u> A polypeptide in bacteroids or in free living cultures. In addition a coding region immediately downstream from <u>nif</u> A hybridized specifically with <u>R.leguminosarum</u> 248 <u>fix</u> Z, a homologue of the <u>K. pneumoniae</u><u>nif</u> B locus involved in FeMo-cofactor production (Rossen <u>et</u><u>al</u> ., 1984).Furthermore, as described in chapter V, a 10 kb <u>nod</u> region has been mapped on the PRE <u>sym</u> -plasmid approximately 7 kb downstream from the <u>nif</u> B homologous gene, based on the homology with an <u>R.meliloti</u> probe carrying nod-genes pRmSL26 (Long <u>et</u><u>al</u> ., 1982). Three different classes of Nod phenotypes, <u>i.e.</u> deficient, reduced and delayed in nodulation, resulted from Tn5 mutations in this region, and confirmed that this region of the <u>sym</u> plasmid contains nodulation genes.Besides the determination of interspecies DNA homologies, we also used an alternative approach for the detection of <u>sym</u> genes. This was based on the observation of Krol (1982) that certain <u>sym</u> -plasmid regions are exclusively transcribed during endosymblosis. In chapters Ill and V we show that screening of a <u>sym</u> -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 <u>nif</u> HDK operon and the <u>nif</u> A - <u>nif</u> B - <u>nod</u> cluster respectively.Subfragments of the two <u>sym</u> -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 <u>fix</u> genes.A contiguous cluster of such <u>fix</u> 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 <u>sym</u> -plasmid resembles the <u>fix</u> ABC operon in <u>R.meliloti</u> , as judged from hybridization data, molecular weights of proteins and the location upstream from <u>nif</u> A - <u>nif</u> B (Ausubel <u>et</u><u>al</u> ., 1985; Weber <u>et</u><u>al</u> ., 1985); the 23 kD <u>fix</u> 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 <u>sym</u> -plasmid of <u>R.leguminosarum</u> PRE. The genetic organization of <u>sym</u> -plasmids has been compared for different rhizobial species; remarkable similarities exist <u>e.g.</u> conservation of the <u>fix</u> ABC - <u>nif</u> A - <u>nif</u> B cluster, but in general the <u>R.leguminosarum</u> PRE <u>sym</u> -plasmid shows larger distances between and shifts in the location of symbiotic genes