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Most R. leguminosarum strains produce bacteriocins which have been traditionally classified as small and medium. based on apparent size (Hirsch, 1979). The small bacteriocin has been identified as a N-acetylated homoserine lactone (Schripsema et al., 1996) but the structure and function of the medium bacteriocins remain uncharacterized. We isolated and began characterization of the medium encoding regions from two different R. leguminosarum strains, bv. viciae 248 and bv. trifola 162Y10. In both cases, sequencing data for the production region demonstrated substantial homology to the calcium-binding regions of RTX toxins such as haemolysin (hly A) and leukotoxin (lkt A). These pore-forming cytolysins are produced by Gram-negative bacterial pathogens such as E. coli and Pasteurella haemolytica (Welch, 1991). Indeed support for a calcium-binding function for the bacteriocin from strain 248 was obtained when the bacteriocin bioassay for this strain demonstrated exogenous calcium-dependence. Even though both 248 and 162Y10 bacteriocins show homology to RTX toxins, cross-hybridization was not observed by Southern analysis under low stringency conditions. In fact, probes from both the 162Y10 and 248 RTX-like regions did not detect homologous determinants in any other medium producing Rhizobium strains tested indicating that these bacteriocins are unique. The 162Y10 bacteriocin also requires an ABC-type transporter for expression which is a novel feature of this strain since it is not found in 248.

This chapter is about the “general genetic knowledge” of the family Rhizobiaceae, Since this “family” is not a true family in any phylogenetic sense, it is very difficult to generalise about the genetics of the group (see Chapter 1). The phylogeny of its members will be discussed elsewhere, but suffice it to say here that the “family” has traditionally been defined in terms of the ability of the bacteria belonging to it to induce the formation of certain types of growths (e.g. nodules, tumours) on plants (Jordan, 1982). Since it has usually been because of this ability to interact with plants that individual members of the “family” have been studied, much information available about the genetics of these organisms is biased towards study of genes involved in symbiotic or pathogenic interactions. It is our intent in this chapter to review the basic genetics of some of the better-studied members of the Rhizobiaceae with an emphasis on tools available for genetic study. Where useful information regarding more “exotic” bacteria is available it will be introduced. The discussion will centre on Rhizobium leguminosarum, Rhizobium (syn. Sinorhizobium) meliloti, Agrobacterium spp. and Bradyrhizobium japonicum.

It is evident that the knowledge about biological nitrogen fixation was enormously increased when genetic systems in free-living and symbiotic nitrogen fixing bacteria were developed and used for the analysis of their nif (nitrogen fixation) genes. Due to its close relationship to Eschericia coli, Klebsiella pneumoniae, a facultatively anaerobic procaryotic microorganism, was initially selected to analyze nif genes (Streicher et al., 1971; Dixon, Postgate, 1971). For other Gram-negative nitrogen fixing microorganisms, genetic systems were recently developed and are now used to compile information in the field of nif genetics. To review all the information available would certainly exceed the scope of this paper. Therefore, we concentrate on three different nitrogen fixing species which are currently being analyzed in our laboratory: the facultative anaerobe Klebsiella pneumoniae, the phototrophic Rhodopseudomonas capsulata and the symbiotic nitrogen fixing Rhizobium meliloti. For these three species, the genetic techniques developed will be outlined. It is our special intention to show that break-throughs are often achieved following the development of new methodology.