J. Gen. Appl. Microbiol., 51, 395–405 (2005)

teria and their relatives,” are a major class of bacteria (Gupta, 2000; Holt et al., 1994; Stackebrandt et al., 1988; Zinder, 1998). This group of predominantly Gram-negative bacteria contains more than 200 genera with a great diversity of phenotypic and physiological attributes. Proteobacteria have been classified based on homology of 16S ribosomal RNA or by hybridization of ribosomal RNA or DNA with 16S and 23S ribosomal RNA (Fox et al., 1980; Woese, 1987; Woese et al., 1985). The class has been divided into five major groups, a-, b-, g-, dand e-. The g-proteobacteria include several families of utmost biological significance, several human, animal and plant pathogens, most notably the Enterobacteriaceae, Legionellaceae, Pasteurellaceae, Pseudomonadaceae, Vibrionaceae, and Xanthomonadaceae. Because of their biological importance, the genomes of more than 33 g-proteobacteria have been fully sequenced and are freely available in GenBank. The 16S ribosomal RNA, the RNA component of the ribosome small subunit, has been established as the macromolecule of choice for single-gene phylogenetic analyses (Lane et al., 1985; Woese, 1987; Woese et al., 1990). The 16S rRNA is an essential component of protein synthesis and is present in all bacteria. Because it is under highly constrained function, its gene is highly conserved throughout bacteria and even very distant bacterial species can be compared. The gene is easy to amplify and sequence using universal primers (Stackebrandt et al., 1991). It is assumed that the homology between 16S rRNA sequences from different bacteria reflects the phylogenetic relationship between these organims. However, the copy number of 16S rRNA genes per bacterial genome ranges between 1 and 15 (Klappenbach et al., 2001). Consequently, one of the premises in 16S rRNA sequenceinferred phylogenies is that a single 16S rRNA allele, any one, is representative of its taxon. It has long been assumed that the 16S rRNA allelic sequences within a bacterial isolate were nearly identical and the homology was governed by concerted evolution (Hillis et al., 1991). In the last few years, however, 16S rRNA sequence heterogeneities have been reported for some bacteria at the intraspecies or intrastrain levels. This is the case for Phormium yellow leaf phytoplasma (Liefting et al., 1996); Mycobacterium strain “X” (Ninet et al., 1996); Paenibacillus polymyxa (Nubel et al., 1996); Study of the heterogeneity of 16S rRNA genes in g-proteobacteria: Implications for phylogenetic analysis