As we described above, some approaches for isolation of specific probes comprise screening of partially described regions of a bacterial genome. Since R. galegae is peculiar in that it nodulates ony a specific legume host,approaches were adapted to locate R. galegae species-specific hybridization probes from the genes determining host specificity. Limited information on the localizaton of these genes was known from previous hybridization experiments.49 Fragments of DNA, carrying the host specificity gene region of R. galegae, were analyzed for specificity in dot blot hybridization experiments.50 The blots contained total DNA from representatives of different rhizobial species and other soil bacteria. One of the probes hybridized strongly with DNAs from all tested R. galegae strains, but showed a weak reaction with two nontarget organisms (Rhizobium loti and Erwinia carotovora). Therefore, smaller sub-fragments of the probe were further analyzed (Fig. 4.1). One of the sub-fragments showed species-specificity: in Southern blot experiments with EcoRI digested total DNA of different bacteria, as it hybridized only with R. galegae DNA. The DNA fragment was sequenced and PCR primers were designed at the ends of it (Fig. 4.1). The specificity of the primers
EcoR Sal\ HindU EcoR
kb kb kb
Fig. 4.1. Fragments of genomic DNA from R. galegae HAMBI 1174, which carry host specificity genes, were analyzed for species specificity. The 2.4 kb EcoRI-fragment shown here was a good candidate but it weakly hybridized with DNA from nontarget organisms. Its three subfragments were also analyzed, and the 0.9 kb EcoRI-SaH fragment was species specific. Arrows show PCR primers.
was verified in PCR reactions with purified genomic DNA from an assortment of strains of R. galegae, other rhizobia and some ether species.51 PCR amplification always produced an 850-bp specific fragment with R. galegae DNA. PCR-RFLP of this species-specific fragment revealed a difference between R. galegae strains according to their host plant G. orientalis and G. officinalis (Fig. 4.2).
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