Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen fixing nodules was investigated by hybridization with RNA from bacteroids. These experiments are described in Chapters 3 and 4. From these experiments it is concluded that only one of the two plasmids present in PRE is strongly expressed in the symbiotic state. On this plasmid also the nitrogenase genes are located. Hooykaas et al . (1981) showed with genetic techniques that a large number, if not all, of the rhizobial genes involved in nodule formation and nitrogen fixation are carried on only one of the plasmids occurring in R. leguminosarum . Therefore they proposed to call such plasmids Sym plasmids, since obviously these plasmids are essential for the development of the symbiotic state. Our results which indicate that only one of the plasmids of R. leguminosarum is strongly expressed in the symbiotic state are in good agreement with the results of Hooykaas et al . (1981). Sym plasmids from different isolates of R. leguminosarum vary remarkably in size, as shows in Chapter 4. The reason why some Sym plasmids contain much more DNA then other Sym plasmids is still unknown. Besides the Sym plasmid a large number of R.leguminosarum strains contain at least one other plasmid, and many strains even more. No function has till now been allocated to these plasmids.For comparison with the hybridizations of plasmid DNA with RNA from bacteroids, plasmid DNA was hybridized with RNA from free living bacteria. No plasmid specific hybridization with RNA from free living bacteria was detectable, as is shown in Chapter 3. A similar result was obtained by Prakash (1981), who could not detect hybridization of plasmid DNA with RNA from exponentially growing bacteria. However, with RNA from stationary bacteria he found hybridization with some restriction fragments of the plasmid. On the physical map of a Sym plasmid which he constructed the part which is expressed in stationary bacteria differs from the parts which are expressed in bacteroids (Prakash, 1981). Both his results and ours indicate that a number of plasmid-borne genes are only expressed in the symbiotic state, and that plasmid-borne genes in exponentially growing bacteria are not expressed, or at the most at a level below detection. It is possible that in exponentially growing bacteria messenger RNA is more rapidly degraded than in bacteroids or in stationary phase bacteria. This could make the detection of specific expression of genes more difficult in exponentially growing bacteria. Rhizobia without Sym plasmids are still viable (Hooykaas et al . , 1981) so expression of this plasmid DNA is not necessary for growth of free living bacteria.Among the plasmid-borne genes which are strongly expressed in the symbiotic state are the nitrogenase genes. This was demonstrated by hybridization of plasmid DNA with plasmid pSA30. Plasmid pSA30 is a recombinant plasmid containing the nitrogenase operon of Klebsiella pneumoniae (Cannon et al ., 1979). pSA30 is only homologous to the part of Rhizobium nif H, coding for the subunits of component II of nitrogenase, and to a part of nif D, coding for the αsubunits of component I (Ruvkun and Ausubel, 1980). It is therefore not possible to determine with pSA30 as a probe in hybridizations with Rhizobium DNA whether nif K, coding for the βsubunits of component I of nitrogenase, constitutes in a single operon together with nif D and nif H, as is the case in Klebsiella pneumoniae (MacNeal et al ., 1978). A decisive answer might be obtained if it would be possible to detect intact messenger RNA from the nitrogenase operon of Rhizobium . As described in Chapter 5 it is possible to isolate large messenger RNAs from bacteroids. Based on the molecular weight of the RNA and on hybridizations with fragments of pSA30 coding for nif H and nif D respectively it is concluded that in Rhizobium nif H, coding for the subunits of component II, is located in a single operon together with nif D and nif K, coding respectively for the αand βsubunits of component I. Bisseling (1980) studied the regulation of the synthesis of the nitrogenase components I and II of R. leguminosarum in pea root nodules. It was shown by radioimmunoassays that component I could be detected 1 or 2 days before component II during the development of the root nodules. During waterlogging component I was still synthesized, whereas the synthesis of component II was repressed. These results indicate that the synthesis of component I and component II of nitrogenase of R. leguminosarum can be regulated rather independently. if the genes for the subunits of the components I and II are transcribed in a single messenger RNA, a regulation on the level of translation of this messenger has to be postulated to explain the different rates of synthesis of component 1 and component II during nodule formation or in waterlogging root nodules. Whether this difference in regulation of the synthesis of component I and component II is on the level of transcription or translation cannot be concluded from our experiments. For that the synthesis of nitrogenase messenger RNA has to be examined under conditions which result in different rates of synthesis of component I and component II. Now the analytical devices are available isolated nitrogen fixing bacteroids may have good potentialities in further study of the mechanism of the regulation of the nitrogenase genes in Rhizobium.